The Canadian Society for Bioengineering La Société Canadienne de Génie The Canadian society for engineering in agricultural, food, Agroalimentaire et de Bioingénierie environmental, and biological systems. La société canadienne de génie agroalimentaire, de la bioingénierie et de l’environnement

Paper No. CSBE18-217

Comparative Fermentation Analysis of Shorts and Clear to Produce Lactic Acid

Kjeld Meereboer1 and Ping Wu2 1. University of Guelph, Guelph, Canada 2. Ontario Ministry of Agriculture, Food and Rural Affairs

Written for presentation at the CSBE/SCGAB 2018 Annual Conference University of Guelph, Guelph, ON 22-25 July 2018

ABSTRACT Wheat shorts and clear flour are by-products generated during flour milling that are either sold as animal feed or added back to certain types of products. The residual carbohydrates make them a suitable carbon source provided appropriate measures are taken to free the fermentable sugars. The objective of the study is to validate the effectiveness of extraction of carbohydrates originating from both wheat shorts and clear flour. The extraction process consists of thermal hydrolysis and enzymatic hydrolysis of wheat shorts and clear flour to free fermentable sugars and take 2 days. The fermentation process is completed at 37°C for 5 days, and in doing so the two by-products can be compared as a carbon source in terms of fermentable sugar and lactic acid yield. Keywords: wheat shorts, clear flour, lactic acid, fermentation, wheat middlings. Keywords: wheat shorts, clear flour, lactic acid, fermentation, wheat middlings.

INTRODUCTION Wheat shorts Wheat shorts and clear flour both originate from milling of wheat, in Canada the most widely grown wheat is Canada Western Red Spring wheat due to its benefits in both milling and baking applications and thus can be a representative source of wheat shorts and clear flour by products in Canada (Canadian Grain Commission 2017). During the milling process shorts, and germ, of which consists of 25-30% the milling process output, is relegated to animal feed. The wheat shorts produced can be defined as a combination of fine particles of wheat bran, germ, and flour (Blasi et al. 1998), as well as residual offal or tailings

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(Bell 2003). Additionally, wheat shorts must not contain more than 7% fiber. The flour milling waste stream contains the majority of protein, minerals, and vitamins of the wheat kernel (Blasi et al. 1998), but wheat shorts by itself provides little in the way of nutrients, consisting of 17-18% protein, of which 70-75% is digestible and poses a poor degree of energy digestibility relative to other animal feed alternatives (Young and King 1981). Hence wheat shorts is usually combined with other flour milling by-products such as wheat germ and a significant portion of wheat bran to make animal feed or wheat middlings (Blasi et al. 1998). Despite wheat shorts having a poor degree of energy digestibility, it still remains a fact that shorts consist of 60% carbohydrates (Phillips Jr. and Balzer Jr. 1957). For experimental comparison, it is assumed wheat shorts is primarily made from wheat bran. The composition of wheat shorts is likely to vary depending on the type of flour produced, thus Table 1 indicates the approximate composition of the primary components that make up the waste stream from flour milling and bran is assumed to be of similar composition (Fraser and Holmes 1959). Furthermore, Tcachuk and Irvine reported wheat bran, , and germ all contain all amino acids (Tkachuk and Irvine 1969), and Fardet reported 2% of wheat bran consists of bioactive compounds, including vitamin B (Fardet 2010). Table 1: Wheat middling’s composition. Endosperm Germ Bran Moisture (%) 14.0 11.7 13.2 Protein (%) 9.6 28.5 14.4 Fat (%) 1.4 10.4 4.7 Ash (%) 0.7 4.5 6.3 Total Carbohydrates (%) 74.1 44.5 60.8 Starch (%) 71.0 14.0 8.6 Hemicellulose (%) 1.8 6.8 26.2 Sugars (%) 1.1 16.2 4.6 Cellulose (%) 0.2 7.5 21.4 Clear flour Clear flour is a co-product of patent flour milling and contains a higher ash content relative to it (Blasi et al. 1998). Clear flour can be further defined as first clear and second clear flour, indicating the degree of ash content, with the latter having the highest (FDA 2003). Similarly, the output of clear flour can vary. Flour can vary from 0.30 to 0.99% ash content during milling runs and largely depends on the type of wheat milled, the type of flour whether it be white or whole wheat, or the final type of flour (Canadian National Millers Association 2017). What’s left with higher ash content, is unsuitable for patent and, Posner and Deyoe considered these to be clear flour (Posner and Deyoe 1986). To a certain extent clear flour can be added back to , but due to its higher ash content and discolouration, it may result in an undesirable final product (Lin et al. 2012). Regardless, it can improve the quality of some flours or be used to produce lower grade grain products such as (Sissions et al. 2008). Table 2 indicates the clear flour composition of wheat, to approximate the composition of clear flour (Sayaslan et al. 2018). Table 2: Durum wheat flour composition. % Composition Moisture 12.5-14.1 Protein 14.4-14.9 Fat 2.1-3.7

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Ash 1.28-1.72 Total Carbohydrates 79.8-82.2 Starch 66.7-70.5 Non-Starch 11.6-13.1 Damaged Starch 4.9-6.2 Lactic Acid Due to the high carbohydrate composition of clear flour and wheat shorts, both hold potential as carbon sources for fermentation in added value processing and lactic acid is a suitable product. 85% of the total lactic acid demand in manufacturing is from the food industry due to its desirable sensory properties over other food grade acids and the preservative functions it can fulfil (Datta et al. 1995). Two percent lactic acid has been reported to be effective as an antimicrobial agent in the meat industry against salmonella species (Killinger et al. 2010), as well as has potential use in industry as a disinfectant for carcasses and equipment (Datta et al. 1995). Additionally, lactic acid can be used as an antimicrobial, curing, pickling and flavouring agents in food (FDA 2017). Lactic acid is also heavily utilized in the development of polylactic acid, a biodegradable polymer, through polycondensation with minimal carbon foot print (Tsuji 2014). With varying processing techniques, desired functionality can be achieved for plastics, textiles and biomedical applications (Avérous 2008). Pre-fermentation Treatment The initial pre-treatment of grain based by products during extraction can be completed in varying manners. The documentation of the extraction of similar products to wheat shorts are more thoroughly covered and significantly more useful in interpreting the ideal method to extract and utilize the potential carbohydrates present in wheat shorts and clear flour. Wheat bran accounts for a considerable portion of milling byproducts. Pre-treatment of wheat bran is a common occurrence to supplement the extraction of carbohydrates upon which enzymes act. Favaro et al. reported heat treatment on wheat bran at 121°C for 30 minutes was effective in exposing carbohydrates to the subsequent enzymatic hydrolysis phases when used with minimal sulphuric acid. Lower temperatures proved ineffective in extracting carbohydrates (Favaro, Basaglia, and Casella 2012). Pre-treatment is likely to assist in enzyme hydrolysis following Manelius at al., who have reported increased enzymatic activity on small starch granules (Manelius et al. 1997). Although neglecting autoclaving procedures in repeated batch processing appears an ideal method to overcome limitations of autoclaving equipment while being less expensive and more desirable concept in industrial scaling and is something intended to be explored (Wang et al. 2017). Tirpanalan et al. utilized a ratio of 1:4, wheat bran to water, and enzymatic hydrolysis was directly implemented using amylase at 85C for 3 hours with a pH of 6.5 and amyloglucosidase at 55°C for 18 hours with a pH of 5.5 and indicate most if not all available starch was effectively hydrolysed into free glucose (Tirpanalan et al. 2015). Other approaches are founded in a similar manner, Favaro et al. utilized a two stage hydrolysis for cellulose, hemicellulose (Favaro, Basaglia, and Casella 2012). Alternative methods can entirely neglect enzymatic, acid and thermal hydrolysis among other methods and rely on the bacterial organism to directly produce lactic acid. Naveena et al. utilized solid state fermentation to directly produce lactic acid in a single step process, effectively reducing the subsequent cost of enzymes, acids and energy. In this case the addition of yeast extract among other additives were deemed necessary to increase production (Naveena et al. 2005). A significant detriment to solid state fermentation is the duration of fermentation was extended up to a week compared to Tirpanalan et al. which took 2 days with a liquid broth, though inoculum preparation of lactobacillus was required (Tirpanalan et al. 2015).

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A latter step that can be considered is the removal of undesireable chemicals of which can increase the ease of purifying the lactic acid. Tirpanalan e al. utilized nano-filtration pre-fermentation to provide a purer substrate for fermentation purposes (Tirpanalan et al. 2015). Brewers Spent Grain In the case of alternative but similar waste streams such as spent brewers grain similar approaches have been made. Brewers spent grain is a product having undergone partial extraction during mashing. Pejin et al. utilized dried spent grain pre-treated with H3PO4 before hydrolysis with alpha-amylase, amyloglucosidase and cellulase at 90°C, 55°C and 45°C respectively (Pejin et al. 2015). Radosavljević et al. used the same extraction method with thin stillage byproduct as an additive over yeast extract as cost saving measures during fermentation (Radosavljević et al. 2018). Lactic Acid Fermentation Lactic acid fermentation in industry is commonly completed using strains of lactobacillus species such as bulgaricus, leichmanii, delbrueckii, amylophilus and plantarum. Although these are only a few, industrially they are favoured over chemical production methods due to more selective stereoisomer production (Pal et al. 2009). Lactic acid fermentation can be completed through batch, continuous and semi-continuous processes. To maximize lactic acid output, an ideal approach is cell immobilization or recycling, involving either entrapment or adsorption, or membrane filtration throughout fermentation. Membrane filtration to remove lactic acid functions by minimizing the impact on the pH (Zhang et al. 2014). Additional substrate can be added in a continuous fashion, maintaining peak lactic acid production throughout fermentation. As cell density increases, fouling of membranes can occur and similarly entrapment may fail due to disruption of the matrix with biomass growth (Senthuran et al. 1997). Traditional lactic acid production utilized calcium hydroxide as pH control, of which functions in multiple facets. A need for lactic acid sequestering is paramount due to its inhibitory effects on lactic acid bacteria growth at high concentrations. Calcium forms calcium lactate in the presence of lactic acid during fermentation, thereby minimizing the effect of lactic acid on the fermentation organism (Demirci et al. 1998). Alternate pH control measures such as calcium carbonate function in a similar manner, by sequestering lactic acid and modulating the pH (Pejin et al. 2015). Lactic acid is recovered through calcium salt precipitation, where the precipitated calcium lactate from fermentation is purified utilizing sulphuric acid. A secondary precipitate, calcium sulphate, forms and is removed, leaving a pure solution of lactic acid (Senthuran et al. 1997).

METHODOLOGY MRS Broth Inoculum The inoculum of lactobacillus acidophilus was prepared using MRS broth from Sigma Aldrich, following the provided instructions, following the suggestion of Tirpanalen et al. of a 1:100 ratio of inoculum to reactor volume (Tirpanalan et al. 2015). The MRS inoculum was autoclaved, and inoculated with 6 billion cfu of L. acidophilus and incubated for 24 hours. Due to water losses in the extraction phase, the actual reactor volume was reduced but the inoculum volume utilized remain unchanged. Extraction A sample of 1kg wheat shorts was added to 10 litres of boiling water in a pressure cooking vessel. The vessel was sealed for fifteen minutes while limiting the peak internal pressure to 103 kPa with an automated release valve to simulate autoclaving and assist in the extraction of carbohydrates in a pre-treatment step, reflecting that of Favaro et al. methodology (Favaro, Basaglia, and Casella 2012). Hydrolysis Enzymatic hydrolysis was implemented after thermal hydrolysis following the design by Tirpanalan et al. with the neglect of the pH modulation to further simplify the process for potential

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industrial scale applications (Tirpanalan et al. 2015). The process involves the addition of 0.5 mL of Termamyl (alpha-amylase) at 85°C for 3 hours and 1 mL AMG 300L (amyloglucosidase) at 55°C for 24 hours to complete enzymatic hydrolysis. Following hydrolysis, the wheat shorts were removed from the fermentation substrate using a filter bag. The resulting solution, termed fermentation substrate, was autoclaved and distributed among 3 glass vessels for fermentation. Fermentation Each autoclaved vessel was inoculated with 33 mL of L. acidophilus inoculum and the pH was adjusted to 5.5 using 10% calcium hydroxide. Fermentation was completed at 37°C and 90 rpm until the pH remain unchanged. Fermentation, now termed fermentation broth, was ended by raising the pH to 10 with calcium hydroxide.

Analysis Lactic acid Lactic acid was measured utilizing the methodology outlined by Borshchevskaya et al. using iron (III) chloride to make the reactant that was used to develop the standard curve of for a 10g/L lactic acid solution (Borshchevskaya et al. 2016). The standard curve was developed from six different known concentrations of lactic acid ranging from 0 to 10 g/L with an R2 value of 0.99 or above when measured at 400 nm. Samples were diluted by 10 and tested in triplicates at the dictated absorbency. Protein Protein assay was completed utilizing the Bio-Rad protein assay dye reagent from Bio-Rad and the bovine serum albumin as a standard to develop a standard curve with an R2 of 0.98 or above using six different known concentrations. The protein assay was diluted by 4 and added to 50 µL samples and the absorbency was measured at 595 nm. Carbohydrates Carbohydrates were tested following the phenol-sulphuric acid assay theory outlined by Cui and Brummer (Cui and Brummer 2005). Samples of the fermentation substrate were filtered with 0.45 µm paper filters. 5 mL of sulphuric acid and 1 mL of phenol was added to 3 mL of sample, after which it was incubated for 30 minutes at 30°C. The absorbency was measured at 490 nm.

RESULTS AND DISCUSSION Carbohydrates Table 3 indicates the extracted carbohydrates from 1 sample of wheat shorts and 2 samples of clear flour after completion of enzymatic hydrolysis. The yield of carbohydrates extracted from clear flour was 47.2% and 42.5% while 15% of the carbohydrates from wheat shorts was extracted. Due to the filtration required for the carbohydrate test, it is expected carbohydrates that remained unhydrolyzed were captured and filtered out, giving a reduced carbohydrate yield of 19.33 g/L. Both samples of clear flour fermentation substrate achieved 59 g/L. Some solids were filtered out of the clear flour sample both before autoclaving and for the carbohydrate test there were not documented, thus the completion of the carbohydrate extraction could not be appropriately assessed because the exact composition of the clear flour received was not analysed. Additionally, the potential for both wheat shorts and clear flour as a carbohydrate source has not been clearly determined with optimizations to the extraction process. Tominaga and Sato reported hydrolysis of saccharified flour based products such as rice flour can be completed in under 24 hrs with 0.0025% glucoamylase with an activity of 300 U/mg (Tominaga and Sato 1996), of which is above that used in this experiment. Furthermore, in an industrial scale, autoclaving is an undesirable feature but is required to ensure initial sterility for lactobacillus. Ouyang et al. have reported using lignocellulosic hydrolysates from corn stover and that fermentation without sterilization may be a feasible alternative that can save both cost and time (Ouyang et al. 2013). Eliminating autoclaving of any sort would require a product free of spores to ensure l. acidophilus is the dominant bacteria. Table 3: Wheat shorts and clear flour carbohydrate extraction data. 5

Trial Carbohydrates (g/L) Yield (g carbohydrates/g substrate) Clear Flour 1 59.733 0.472 Clear Flour 2 59.033 0.425 Wheat Shorts 2 19.333 0.145 The results are indicative of the potential as a lactic acid bacteria feed source clear flour is when comparing the yield to the starch content present in Durum clear flour of Table 2. The available starch extracted from wheat shorts is indicated in Table 4 where the remaining starch was reduced to below 1%. Due to solid losses from extraction, to determine the exact degree of completion for starch extraction becomes complicated. Regardless, lower yields of carbohydrate extraction can’t be considered a completely negative effect as wheat shorts can still be utilized as animal feed after extraction much like brewers sent grain and are valued based on their nutritional content and protein. Table 4: Nutritional composition of wheat shorts data.

Book Values - Raw Shorts Spent Shorts Nutrient Wheat middlings Average Value Average Value AF AF AF DM (%) 88.85 88.997 28.307 Moisture (%) 11.15 11.003 71.693 Protein (%) 18.56 17.89 6.42 Soluble Protein (%) 6.7 0.685 Protein Soluble Protein as % CP 40.48 NDF-CP (NDP) (%) 0.68 0.67 NDF-CP (NDP) (%) 5.2 6.79 Crude Fibre (%) 3.45 4.72 ADF (%) 13.23 12.49 6.22 Fibres Neutral Detergent Fibre (%) 38.33 37 18.5 Lignin (%) 3.66 4.67 3.335 Fat (%) -EE 4.14 3.47 1.2 Non-Fibres Starch (%) 25.56 15.27 0.57 Non-fibre carbohydrates (%) 30.075 7.31 Ash (%) 8.15 5.257 1.3 Calcium (%) 0.12 0.053 0.023 Phosphorus (%) 1.08 1.207 0.307 Potassium (%) 1.15 1.137 0.163 Minerals Magnesium (%) 0.43 0.507 0.163 Sodium (%) 0.05 0.02 0.01 Copper (ppm) 12.85 12.59 3.825 Iron (ppm) 144.23 148.2 56.11

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Manganese (ppm) 136.62 119.8 41.795 Zinc (ppm) 87.85 100.7 38.965 TDN (%) 72.9 67.6 19.75

Energy (ADF Net Engery {lac} (Mcal/kg) 1.55 0.425 Based) Net Energy {gain} (Mcal/kg) 1.11 1.185 0.325 Net Energy {maint} (Mcal/kg) 1.73 1.76 0.495 WTDN (%) 60.94 16.645

Energy WNEL (Mcal/kg) 1.38 0.37 (OARDC) WNEG (Mcal/kg) 0.95 0.235 WNEM (Mcal/kg) 1.49 0.4 Energy (swine) Swime ME (Mcal/kg) 2.9 0.92 Titratable acidity Figure 1 and Figure 2 illustrate the titratable acidity as a measure of lactic acid produced throughout the duration of fermentation. Trial 1 sample 3 of the clear flour trial resulted in undesirable exposure of the sterilized sample before inoculation and was thus excluded from the trial. The resulting duration of the clear flour trial lasted longer than the wheat shorts trials with a higher lactic acid concentration by titratable acidity. Figure 1 and Figure 2 both don’t illustrate a notable indication of an exponential phase despite its length which is likely a limitation of the titration method for the trial. To maintain temperature, an incubator was utilized, and more appropriate pH management was unfeasible as a result. Additionally, alternate designs can be used to increase fermentation efficiency. Zhang et al. reported integration of membrane filtration to remove lactic acid and recover biomass for later fermentations increases efficiency by organism acclimation (Zhang et al. 2014).

Figure 1: Lactic acid by titratable acidity of clear flour during fermentation.

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Figure 2: Lactic acid by titratable acidity of wheat shorts during fermentation. The final duration indicated by titratable acidity for clear flour being longer than wheat shorts in addition to producing a higher concentration of lactic acid by the end of the fermentation reflects the information in Table 1 and Table 2. The clear flour is expected to have a higher carbohydrate composition in addition to a larger proportion being simple sugars in the form of starch. Similarly, the lactic acid indicated by titratable acidity reflects that found in Table 3 except the second wheat shorts carbohydrate sample was lower than what the titratable acidity measure indicated. No carbohydrate sample was taken for the first wheat shorts test at the time. Lactic acid Clear flour fermentation produced an average concentration of 52.5 g/L and yield of 42.6% indicated in Table 5. Wheat shorts performed significantly poorly relative to clear flour in terms of lactic acid produced. Table 6 indicates an average concentration of 23.0 g/L and yield of 17.8%. The results reflect the larger presence of enzymatically hydrolysable starch based polysaccharides in clear flour. All lactic acid samples are higher than what the titrable acidity indicates. Table 5: Clear flour lactic acid and protein data.

Lactic Acid Conc. Yield Yield Trial Sample (g/L) (g lactic acid/g clear flour) (g protein/g clear flour) 1 54.061 0.425 Trial 1 0.004 2 51.665 0.399 1 51.267 0.425 Trial 2 2 47.791 0.405 0.002 3 57.782 0.475 Table 6: Wheat shorts lactic acid and protein data. Yield Lactic Acid Conc. Yield Trial Sample (g lactic acid/g wheat (g/L) (g protein/g wheat shorts) shorts) Trial 1 1 23.652 0.178 1.00E-03

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2 21.305 0.160 3 22.433 0.168 1 25.326 0.198 Trial 2 2 25.235 0.200 0.008 3 20.296 0.165 Overall the lactic acid concentration produced from wheat shorts is lower than what Clement et al. reported with the same extraction and fermentation method of which may be the result of variations in the quality of the wheat shorts utilized (Clement et al. 2017). Wheat shorts is a waste stream as mentioned previously, and is a product of producing a type of flour. Variability can be introduced based on wheat breed, variety, and flour milling procedure (Blasi et al. 1998). Protein Protein extraction was minimalized due to the lack of proteolytic enzymes. Pejin et al. documented the extraction of protein from spent grain without the use of proteolytic enzymes had minimal effect (Pejin et al. 2017). With a reduced degree of protein extraction and proteolytic enzymes, deprivation of a more suitable nitrogen source may have impacted the growth of l. acidophilus, resulting in no clear exponential phase. The overall protein has a significant impact on the quality of the post extraction wheat shorts. Table 4 indicates the as-fed (AF) quality of wheat shorts and spent wheat shorts relative to approximated values of wheat shorts, assumed to be a combination of wheat middlings and wheat bran (National Academies of Sciences and Medicine 2016). An 11.47% reduction in crude protein is indicated, a large portion being the soluble proteins. The usage of spent clear flour as animal feed has not been assessed, and likely more vigorous extraction methods may be more suitable to extract and hydrolyze all the starch and protein for bacterial growth and fermentation. Wheat shorts after extraction The total digestible nutrients of wheat shorts was reduced by 47.8% after extraction processes when referring to Table 4. Although this is not considered an accurate indication of the quality of the product, it would need to be considered if spent wheat shorts is to be used as animal feed as is (Preston 2017). The significant reduction in spent wheat shorts quality is the result of both the extraction of nutrients and the increased moisture content. 71.6% of the spent shorts consists of water from the initial 11%. Pre-treatment can be completed to remove the additional water and increase the quality. The significant increase results in a great impact on the value of wheat shorts. Utilizing the Petersen equation, the approximate value of wheat shorts before extraction was $240 and spent wheat shorts were valued at $80.

CONCLUSION Clear flour is indicated to be a more suitable carbon source than wheat shorts for fermentation of lactic acid with l. acidophilus, achieving an average lactic acid concentration of 52.5 g/L over 23.0 g/L from wheat shorts. The carbohydrate concentration from clear flour extraction reflects the lactic acid concentration at 59 g/L. The wheat shorts carbohydrate composition was lower than the final lactic acid concentration at 19 g/L, likely the result of the filtration of unhydrolyzed starches form the carbohydrate test. The yield of lactic acid relative to the substrate for clear flour is twice that of wheat shorts. The higher concentration of available carbohydrates from clear flour also increased the fermentation time of which may be the result of limited free nitrogen due to low protein extraction or the absence of continuous pH management. The reduced extraction in terms of wheat shorts may be more desirable if it is intended to be used as animal feed after the fact. The value of wheat shorts from a nutrient perspective was reduced from

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$240 to $80. Clear flour has not been considered as animal feed, but due to the high starch composition, complete extraction is a more desirable goal.

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