Improved Nutritional Value of Surplus Bread and Perennial Ryegrass Extracts Via Solid-State Fermentation (SSF) with Rhizopus oligosporus

Improved Nutritional Value of Surplus Bread and Perennial Ryegrass Extracts Via Solid-State Fermentation (SSF) with Rhizopus oligosporus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Improved Nutritional Value of Surplus Bread and Perennial Ryegrass Extracts Via Solid-State Fermentation (SSF) with Rhizopus oligosporus David Bryant, Juan Sandoval, Joe Gallagher, Julia Rodriguez-Garcia, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3973183/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Nov, 2024 Read the published version in npj Science of Food → Version 1 posted 10 You are reading this latest preprint version Abstract Solid-state fermentation (SSF) is a sustainable method to convert food waste and plant biomass into novel foods for human consumption. Surplus bread crusts (BC) have the structural capacity to serve as SSF scaffold, and their nutritional value could be increased in combination with perennial ryegrass (PRG), a biorefining feedstock with high-quality protein but an unpleasant sensory profile. SSF with Rhizopus oligosporus was investigated with these substrates to determine if the overall nutritional value could be increased. The BC-PRG SSFs were conducted for up to 72 h, over which time the starch content had decreased by up to 89.6%, the crude protein content increased by up to 113.1%, and the essential amino acid content increased by up to 54.5%. The BC-PRG SSF demonstrated that this process could potentially valorise BC and PRG, both widely available but underexplored substrates, offering feedstock for alternative protein sources. Biological sciences/Microbiology/Applied microbiology Biological sciences/Biochemistry/Proteins Figures Figure 1 Figure 2 Figure 3 Introduction Wheat ( Triticum aestivum ) is one of the most consumed crops worldwide, contributing to 20% of the calories and proteins in human diets 1 , and is the basis of multiple staple foods across different cultures, such as breads, cakes, biscuits, cookies and crackers. Approximately 760 million tons of wheat flour are produced yearly worldwide 2 , of which 10% is wasted 3 . Bakery waste, particularly in the form of bread, is a growing global concern, as 270 million tons of bakery products were produced in 2015, of which 69 million tons were wasted. Some of this waste occurs at the household level, mainly due to spoilage, but also at the manufacture and retail level, where bread waste is called surplus and is primarily comprised of bread crusts and breadcrumbs. While most of this surplus is safe for consumption, it is unsuitable for commercialisation, as it does not comply with the quality specifications given by the manufacturer to the relevant legal authorities, such as the UK Food Standards Agency (FSA), regarding physicochemical composition, and stablished by the manufacturer for appearance and sensory characteristics. The valorisation of surplus bread has been widely explored. Traditionally, it is either milled to be reincorporated into the production of new batches of bread or other products such as soups and fried foods 4 or as an animal feed 5 , but due to the volume of waste generated not all of it is given proper use and ends up being discarded. Ben Rejeb, et al. 6 reviewed the available routes for the valorisation of bread waste into value-added products, both through chemical processes and fermentation, including the manufacturing of ethanol, lactic acid, succinic acid, biohydrogen, hydroxymethylfurfural, isolated proteins, pigments, sugar syrups, aromatic compounds, and enzymes. More recently, solid-state fermentation (SSF) has also been explored as an alternative valorisation process, as bread possesses most of the nutrients needed by SSF microorganisms, specifically filamentous fungi 7 , and provides a structural matrix of gluten, the main protein of wheat, and gelatinised starch in which they can grow 8 . SSF has been used to nutritionally enhance the biomass of cereals, legumes, fruits and vegetables for human consumption 9 , with a low environmental impact. The lack of a liquid phase reduces the generated wastewater and other potential pollutants 10 , while the use of waste materials as substrates minimises the cost of media preparation and reduces the energy consumption of the process, as sterilisation is not commonly required 7 . Amongst the multiple filamentous fungi reported in SSF in the food industry, Rhizopus microsporus var. oligosporus (Rmo) has been used for centuries in the production of tempeh from soybeans ( Glycine max ) 11 . Rmo has been shown to valorise surplus bread, reducing the concentration of starch (from 65.8–42.7%) while increasing the concentration of crude protein (from 13.2–16.1%) and crude fibres (from 11.6–26.7%) 12 and to increase the relative ratio of essential amino acids (AA), minerals and vitamins 13 . Rmo , like other filamentous fungi, can produce proteases that cleave protein, thereby altering the AA profile and increasing the concentration of essential AA 14 , improving the protein quality. However, as Rmo is incapable of nitrogen fixation, increasing the total nitrogen content in the substrate requires external supplementation. Alternative protein sources with high unexploited potential for human applications are forage grasses and clovers. These crops have been studied as a source of fibres and cellulose 15 , fertilisers 16 , biogas and bioethanol 17 , and lactic acid 18 as well as for direct human consumption 19 , with its primary use being animal feed. These crops are rich in high quality protein, and the mineral, i.e., magnesium, potassium, phosphorus and calcium 20 , 21 and nutraceutical content, i.e., fructan, pinitol, isoflavones and tannins 22 , also contribute to their nutritional profile. They can achieve high biomass yields and protein outputs; for instance a square hectare of land can produce between 1.8 and 3 tons of alfalfa protein while the same landmass only 150 to 200 kg of meat protein 23 , with low environmental footprints 24 , which makes them widely grown worldwide. Of these crops, PRG ( Lolium perenne) is one of the most used grass species in the world, especially in Europe where it represents up to 50% of the grass seed market 25 . It’s mainly used as feed in grazing livestock for animal protein production, which compared to using its protein for direct food applications, has been reported to be an inefficient use of the land 26 . It contains a crude protein (CP) content of approximately 12% (dry matter (DM)) and a similarly well-balanced amino acid profile as soybean 27 , due to the composition of RuBisCO (ribulose-1,5-bisphosphate carboxylase-oxygenase), a key enzyme in the fixation of CO 2 during photosynthesis, which makes up to 50% of the total soluble protein content 28 . Despite this, PRG is not adequate for direct human consumption due to its high content of undigestible fibres, particularly cellulose, which can range between 30% and 50% DM 29 , and its unpalatability. Extraction, fractionation, purification and/or chemical and biological conversion of the protein in forage crops is required to make use of it in food applications and has been widely explored by different methods, although with an emphasis on animal feed 28 . Thus, this research project proposes a novel sustainable process to valorise surplus bread crusts and PRG biomass, a combination of substrates reported here for the first time in the literature. It is hypothesized that the protein content and the concentration of available essential amino acids in the final product will increase by combining these substrates via SSF with Rmo . This work provides a fundamental study for the application of forage crop proteins in producing an alternative source of high-quality protein for human consumption via SSF, which would provide useful information for future developments in novel foods. Results and discussion Solid-State fermentation Surplus bread crusts (BC) are a relatively dry material (79% DM) with a CP content of 16.3 ± 0.2% DM, crude fibre content of 15.4 ± 0.2% DM and starch content of 69.8 ± 0.3% DM that can be transformed into an alternative food via SSF as part of a strategy in the management of food waste. The porosity of the structure makes the nutrients accessible for Rmo , which allows the mycelia to grow throughout the whole substrate and not just superficially, as has been seen in soybeans where the mycelium can penetrate approximately 2 mm into the substrate within 40 h of fermentation 30 . Exploiting the structure and dimensions of BC was fundamental in accelerating the growth of Rmo. Firstly, the use of 1x1x1 cm BC cubes allowed the fungi to grow throughout the substrate (as sown in Figure S1 ), in contrast to SSF with milled BC (as shown in Figure S2 ) which only supported superficial growth as Rmo could not penetrate the substrate. Secondly, the initial substrate moisture of 56% was optimised from a range of 40 to 70% to the maximum consumption of starch after 72 h of fermentation (as shown in Figure S3 ). Thirdly, a pH of 3.5 was selected to avoid the growth of undesirable microorganisms, which was proven to not significantly affect the growth of Rmo (as shown in Figure S4 ). This adjustment was significant due to the addition of grass juice (GJ), a fresh screw-pressed juice of PRG which contained microorganisms that could influence the process. GJ and dry green solids (GS) were added to increase the nutritional value of the SSF substrate in an environmentally sustainable way, as these materials had CP contents of 16.0 ± 0.7% DM and 34.9 ± 0.3% DM, respectively. The first section of experiments, comparing BC/W and BC/GJ, were conducted to provide a baseline of the SSF process and understand the influence of the plant matrix on Rmo (Fig. 1 ). In contrast, the second section, which compared increasing amounts of GS to increase the CP content, were performed to evaluate alterations in the amino acid profile of the protein after the fermentation (Table 2 ). The SSF experimental designs were photographed every 24 h from start to end of the fermentation to record the growth of Rmo over the different BC substrates (Fig. 1 ). Increasing amounts of PRG protein were added to BC (19%, 20%, 23%, 27% or 29% DM) (Fig. 1 I, c-g), and this consistently slowed down the growth of Rmo mycelium. After 24 h of SSF and up to 20% DM CP BC/GJ/GS (Fig. 1 I, d), the fungi was able to superficially cover the whole dish. However, higher quantities of GS (BC/GJ/GS to CP 23%, 27% or 29% DM) reduced this growth (Fig. 1 I, e, f and g, respectively). This deceleration could possibly be a consequence of phenolic compounds in PRG, which, while not as widely studied as in other crops such as red and white clover, have shown to contain organic acids such as gallic acid and protocatechuic acid and flavonoids such as quercetin and kaempferol 31 , all of which have been proven to have antifungal activity at concentrations over 0.3 mg/mL 32 . These concentrations are considerably higher than those naturally found in whole PRG and even less so in GJ, which could explain why inhibition of growth was only evident in higher concentrations of GS. After 48 h, Rmo mycelium had completely covered all samples up to BC/GJ/GS to CP 27% DM (Fig. 1 I, a-f), with BC/GJ/GS to CP 29% DM (Fig. 1 I, g) being partially covered, and after 72 h the fungi started to senesce as noted by the change in colour from white-light grey to yellow-dark grey. This timeframe aligns with the ageing of Rmo in SSF and implies the start of the deterioration of the mycelium, the degradation of the nutritional value and negative changes to the sensory profile of the substrate 30 . Figure 1 (II) details the percentage loss of total substrate mass at 24, 48 and 72 h versus the initial mass at 0 h as an approximate quantitative measure of the growth of the fungi during SSF. A fraction of the mass lost was due to the evaporation of water, while the remainder was attributed to carbohydrate metabolism of the fungi and the production of CO 2 , although the extent by which each contributes to the mass change was not measured. A mathematical model developed by Figueroa-Montero, et al. 33 in a SFF with Aspergillus niger shows the complexity of the mass transfer phenomena in SSF systems, where water and CO 2 are the main outputs of the fermentation. The mass loss of BC/GJ and BC/W (Fig. 1 II, a) was statistically different across time (p = 0.00) and between experimental treatments (p = 0.00) at 24 and 72 h, although no interaction was found between the factors (p = 0.16) ( Table S1 ), with BC/W having a 49.4% higher loss at 24 h and 14.0% higher loss at 72 h; however, no significant differences in mass loss were observed at 48 h (p = 0.08). The mass loss of BC/GJ at different levels of GS (Fig. 1 II, b) had statistical differences across time (p = 0.00) and the different experimental treatments (p = 0.00), but no interaction effect was identified (p = 0.19) ( Table S2 ), with the highest losses generally occurring at the treatments with 19% and 29% DM and the lowest losses with 23% and 27% DM. Chemical composition analysis The chemical composition of the substrates was assessed to evaluate the change in nutritional properties over the course of the SSF (Fig. 2 ). The DM content of all experimental units started at 45.7 ± 0.4% and ended at 43.5 ± 1.3%, without substantial differences at any time point. Although the ambient moisture in the SSF chamber was not controlled, the DM was stable throughout the process. The main driver for the mass change in all macronutrients was the digestion of starch as the primary carbon source of the substrate. These changes have also been described in the literature, as fermentation with Rmo transforms carbohydrates into fungal biomass, thereby increasing the concentration of crude protein and crude fibre 34 . Additionally, the ratio of crude protein to starch content was calculated to help visualise the changes in the chemical composition in terms of the nutritional value of the substrate (Fig. 3 ). Comparing the composition of BC/GJ and BC/W (Fig. 2 a), the nutritional value of BC improved considerably in both cases, and few differences were identified. Crude protein content was equal between the experimental treatments (p = 0.66) at each time point, but it significantly increased (p = 0.00) over time, and no significant interaction was observed between factors (p = 0.38) ( Table S1 ). Analysing the change of both treatments over time ( Figure S5 ) shows an increase in the crude protein content of 74.6% after 72 h of SSF. Crude fibre content was significantly higher (p = 0.01) for BC/GJ at 72 h, likely due to the faster carbohydrate metabolism in BC/W that allowed the fungi to use the fibres as the secondary carbon source. After 72 h, crude fibre increased by 54.2% in BC/GJ and by 24.8% in BC/W. Starch content was significantly lower (p = 0.00) for BC/W at 24 h (Fig. 1 II, a) due to a faster metabolic activity during the first hours of the fermentation, as seen by the higher free sugar content at the same time point as a result of the enzymatic hydrolysis of starch into smaller oligosaccharides and monosaccharides. After 48 and 72 h, the free sugar content is reduced as monosaccharides are used as the main carbon source for fungal growth and transformed into water and CO 2 . After 72 h, the starch content is reduced by 76.8% in BC/GJ and by 76.5% in BC/W. Ash content was significantly higher (p = 0.00) for BC/GJ in all time points likely due to the added minerals, such as phosphorus, potassium, calcium and magnesium, present in GJ, as reported previously in the literature 35 . At the end of the SSF, the ratio of CP to starch increased from 0.3 to 2.3 (Fig. 3 a) which, similar to the changes in crude protein, was significantly different (p = 0.00) across time but not significantly different (p = 0.39) between the experimental treatments ( Table S1 ). As a whole, these results indicated the feasibility of adding forage crop extracts to the SSF process and led to the increasing levels of forage crop protein of the next set of experiments. These changes induced modifications to the flavour profile of the substrates, particularly in BC/GJ, as the material developed sweet notes reminiscent of pineapple and lost all notes related to the green smell of grass, as it was corroborated in informal sensory tests. After 72 h, the senescence of Rmo (Fig. 1 I) developed undesirable pungent smells akin to the growth of the black bread mould R. stolonifer , the most common household bread mould. SPME GC-MS olfactory analysis was carried out over SSF samples to corroborate these findings, but a complete analysis was outside the scope of this publication. Another manuscript detailing these findings is under preparation. The composition of the BC/GJ/GS samples (Fig. 2 b) was influenced by the amount of GS added, as higher GS content resulted in a lower concentration of starch and a higher concentration of CP in the substrate, while maintaining the same initial moisture. This led to statistical differences for all components (p < 0.05) between experimental treatments and across time, except for crude fibre between the experimental treatments (p = 0.20), and significant interactions (p < 0.05) between the factors for crude protein, free sugars and starch ( Table S2 ). As the starch content decreased to 10.3 ± 0.8% DM after 72 h from initial contents of 51.1 ± 1.1% DM to 15.6 ± 1.7% DM at 0 h, the rest of the components were concentrated. After 72 h, only crude protein and free sugar content were statistically different (p 0.05). At this point, crude protein content increased by 67.3% in BC/GJ/GS CP 19% DM. Higher GS levels resulted in lower CP content increases after SSF, achieving a 17.8% increase at BC/GJ/GS CP 29% DM. The ratio of CP to starch content was significantly higher (p = 0.00) in BC/GJ/GS CP 27% and 29% DM after 72 h (Fig. 3 b) than in the other treatments with GS. As with the BC/W and BC/GJ experiments, the change in the free sugar content also induced changes to the substrate aroma, reducing the characteristic grassy smell, as noticed after informal testing. At 72h the CP composition of all the fermented substrates were statistically higher (p = 0.00) than those of unfermented BC (Fig. 2 a, time 0, BC + W) and had a maximum increase in CP of 113.1% in BC/GJ/GS CP 27% DM mirrored by a significant decrease in starch content (p = 0.00) of 89.6% ( Table S3 ). Amino acid profile analysis Essential (EAA) and non-essential (NEAA) amino acids were analysed by HPLC to evaluate the transformation of the amino acid profile of the BC substrates over time. All EAA, except for tryptophan, were quantified and their concentrations are shown as a coefficient of the crude protein content (g of amino acid / 100 g of CP), as well as the sum of the total amino acids (AA), sum of EAA, sum of NEAA, the percentage of EAA over total AA and NEAA over total AA (Table 1 and Table 2 ). The biotransformation of the protein of the substrate, as an effect of the metabolism of Rmo during the SSF, led to improved amino acid profiles. In all experimental units, although to a lesser extent in the GS experiments due to the lower percent composition of BC in the substrates, glutamic acid was the AA with the highest concentration at 0 h (from 11.81 to 27.03 g/100 g CP), as this is the predominant AA in wheat protein. Whilst this AA contributes to a pleasing savoury flavour, it is not nutritionally desirable as it is a NEAA. Coupled with the low concentration of lysine versus the FAO nutritional guidelines 36 , wheat protein as a whole has a low protein digestibility-corrected amino acid score (PDCAAS) of 0.42 37 and thus not considered a high quality protein for human nutrition. The positive effect of fungal metabolism over the AA profile can be seen in the BC/W SSF (Table 1 ) with the AA concentrations that were significantly affected by time ( Table S1 ). The percentage of EAA over the total AA increased from 30.63 ± 1.5% to 36.15 ± 1.5%, a significant 18.0% increase (p = 0.00) after 72 h. At this latter time point, the AA with the greatest significant increases (p = 0.00) over time were threonine (19.3%), alanine (112.9%) and aspartic acid (91.4%) while the AA with the greatest significant decreases (p = 0.00) were glutamic acid (-47.4%) and proline (-38.1%) ( Table S1 ). The bioconversion of certain NEAA, such as glutamic acid, has been studied via metabolic pathways such as the \(\alpha\) -aminoadipate pathway, where glutamic acid is the primary amine source for the production of lysine, which selectively allows fungal metabolism to increase the composition of EAA by depleting NEAA 38 . The variation in total AA was not significant (p = 0.38) over time, but its tendency to decrease is potentially due to the continuous cleaving of substrate protein from fungal proteases as time progresses ( Table S1 ). Decreasing AA as a percentage of crude protein implies a reduction in the actual functional protein, thus just increasing the value of CP is not enough to consider the valorisation of the substrate. Comparing BC/GJ with BC/W (Table 1 ) yields few differences in the AA profile. The relatively slower fermentation of BC/GJ (Fig. 1 ) produces a maximum percentage of EAA at 48 h (36.4%), instead of 72 h (34.0%) as in BC/W, but the total sum of AA is otherwise similar (p = 0.07). Comparing the contents of AA after 72 h of BC/GJ with those of BC/W, BC/GJ had a higher content of phenylalanine, threonine, valine, alanine, glycine, proline, serine, and tyrosine (Table 1 ). Significant differences that show the effect of forage protein over the AA profile can be seen in the experiments with BC/GJ/GS (Table 2 ). Increasing additions of GS not only reduces the glutamic acid content at 0 h, but also reduces proline and increases all EAA. Of particular relevance to the nutritional quality of BC, the increase of isoleucine, leucine, lysine and methionine, all present in L. perenne 27 , greatly covers the AA deficiencies of wheat. Also, at 0 h, the sum of total AA increased from 67.03 ± 7.2 at the lowest level of GS up to 99.05 ± 0.7 g/100 g CP at the highest, a 47.8% increase, and the percentage of EAA increased from 28.72 ± 1.4 up to 42.69 ± 0.0%, a 48.6% increase. After 72 h of SSF, the sum of total AA significantly decreased (p = 0.00) in all experiments between 4.7 and 27.8%, primarily due to the decrease of NEAA, while the percentage of EAA increased by 48.6% at the lowest level of GS and decreased by 8.3% at the highest level of GS. While all EAA were affected throughout the SSF by the addition of GS (Table 2 ), only histidine, lysine, phenylalanine and threonine were affected by the interaction of both the addition of GS and time (p < 0.05), showing the specific metabolic activity of Rmo towards certain AA of interest, such as lysine ( Table S2 ). At 72 h the AA profile of the substrates with added GS were significantly different (p = 0.00) than those of unfermented BC ( Table S3 ). The SSF with BC/GJ/GS CP 29% DM had the highest sum of total AA, a 23.4% increase in comparison to unfermented BC, while the SSF with BC/GJ/GS CP 27% DM had the highest percentage composition of EAA, a 54.5% increase in comparison to unfermented BC. The change of each AA after the SSF varied considerably between each level of GS, likely in response to complex shifts in the metabolic pathways taken by Rmo during SSF, but local maxima and minima can be identified. For instance, the greatest increase in histidine occurs in BC/GJ/GS CP 23% DM (115.4%), of lysine in CP 29% DM (77.8%) and of threonine in CP 19% DM (32.9%), which opens the possibility of targeting the concentration of particular AA at different substrate compositions. Alongside the changes in the proximal composition (Fig. 2 b), this shows that supplementing the CP content of BC with GS results in similar levels of CP (32.5 ± 0.5%) after 72 h of SSF while increasing both the composition of total AA and the percentage composition of EAA. The impact of these changes for food applications can be further seen in Table 3 , which shows the EAA reference ratios calculated against the FAO nutritional guidelines for adults 36 . Currently, calculation of the nutritional quality of protein is performed via the PDCAAS method, which multiplies the lowest reference ratio from the EAA list with the in-vitro digestibility of the protein. This method undervalues proteins that are deficient in particular AA, such as lysine in wheat, and ignores AA far in excess of the reference. The reference ratios of the EAA in the SSF substrates after 72 h of fermentation increase compared to those of unfermented BC, with the exception of isoleucine, leucine and lysine in BC/GJ/GS 19–27% DM CP and of methionine + cysteine in BC/W. As neither tryptophan nor the changes in the digestibility of the protein were quantified a PDCAAS could not be calculated, but the ratios suggest that BC/GJ/GS 29% DM CP would have the highest value, as the lowest reference ratio is still for lysine, but is of 0.88 compared to the 0.65 of unfermented BC. In this same SSF sample, all other reference ratios are over 1.0, with particularly high increments in ratio for histidine (2.78) and phenylalanine + tyrosine (3.97). Reports of the changes in the protein digestibility after SSF with Rhizopus sp. are mixed in the literature 39 , 40 , and could be affected by the phenolic content of PRG and the changes in the free amino acid content of the substrate after SSF. This research establishes the viability and nutritional potential of combining forage crops and bakery surplus to reduce waste, improve circularity within the food industry and upgrade non-conventional crops for further development as human food. As such, this provides novel insights for the potential of other combinations of substrates to be fermented with Rmo , or other food-safe filamentous fungi used in SSF, such as Aspergillus sp. and Neurospora sp. and explore the changes in the nutritional quality and sensory profile. Industrially, this provides the agricultural sector an additional route to valorise their crops through a biorefinery, and the food manufacturing sector information to develop novel alternatives for animal proteins with lower environmental impacts, which would reduce food waste, increase food security, and decarbonize the food system. The resulting fermented products could thus be explored as novel foods, before being dried and milled, or the processed protein-rich powder incorporated in the preparation of wheat-based staple foods such as bread, cookies, pasta, pastries and cakes, by replacing fractions of wheat flour, which requires further research. Materials and methods Raw materials Acetonitrile, formic acid, ammonium formate, ethanol and sodium hydroxide were purchased from Fischer Scientific UK Ltd (Leicestershire, UK). Phosphoric acid, phenol, DL-norvaline, potato dextrose agar and tween 80 were purchased from Merck Life Science UK Ltd (Dorset, UK). Anhydrous acetonitrile, hydrochloric acid, borate buffer and amino acid standard H were purchased from ThermoFischer Scientific Ltd (Paisley, UK). 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was purchased from Synchem UG & Co. KG (Altenburg, Germany). Total starch analysis kit was purchased from Megazyme Ltd (Wicklow, Ireland). Unless specified, all this study's chemicals, solvents and reagents were of at least analytical grade. For HPLC, the AccQ-Tag Amino acids C18 column was purchased from Waters Ltd (Wilmslow, UK), and the REZEX-ROA Organic acid H+ (8%) Ion exclusion column was purchased from Phenomenex Inc (CA, US). Bread crusts (BC) from surplus wheat flour loaf bread were provided by Bradgate Bakery (a division of Samworth Brothers Ltd, Leicestershire, UK) and manually cut to approximately 1x1x1 cm cubes. Perennial ryegrass harvesting and pilot-scale processing Perennial ryegrass ( Lolium perenne ) was seeded in 2021 in a farmed experimental plot at Aberystwyth University in Aberystwyth, UK and harvested in June 2022. 1 ton of freshly harvested PRG was screw pressed at pilot scale in a CP-10 screw press (Vincent corporation, FL, US) to obtain 410 L of GJ with a pH of 5.62 (adjusted to pH 3.5 with H 3 PO 4 ) and frozen at -20°C in 2 L aliquots to avoid the growth of undesired microorganisms. The resulting GJ had a DM content of 4.98% and CP content of 16.0% DM. 32 L of GJ were clarified using a GLE continuous centrifuge (Carl Padberg Zemtrofugenbau GmbH, Lahr, Germany) at 38000 x g to obtain 800 g of wet protein-rich precipitate with a DM content of 27.3%, which was freeze-dried to a GS with final DM content of 85.0% and CP content of 34.9% DM. Microorganism and inoculum preparation Rmo spores were purchased from fermentationculture.eu and preserved in 10% glycerol solution at -80°C and in potato dextrose agar (PDA) plates at 32°C. After 5 days of growth at 32°C Rmo spores were harvested from PDA plates in 50 mL sterile distilled water (0.03% tween 80) and counted in a Neubauer chamber to approximately \(1\times {10}^{7}\) spores/mL and SSFs were inoculated at \(2\times {10}^{5}\) spores/g substrate. Solid-state fermentation SSF experiments were conducted in sterile 100 mm diameter Petri dishes. The experiments were divided into two sequential sections: firstly, 25 g of BC were adjusted to a moisture content of 56% (v/w DM) with either sterile distilled water (W) or GJ and fermented for 24, 48 or 72 h. These experiments were done in triplicate for a total of n = 24 samples. Secondly, 25 g of BC were adjusted to a moisture content of 56% (v/w DM) with GJ, supplemented with GS to final substrate CP contents of 19%, 20%, 23%, 27% or 29% DM, and fermented for 72 h. These experiments were done in duplicate for a total of n = 24 samples. For each fermentation, 40 g of mixed substrate (BC with W/GJ/GS), at pH 3.5 (adjusted with H 3 PO 4 ), covered with lids but not closed hermetically to enable airflow, were incubated at 32°C, having been weighed and photographed before and after the SSF. All fermented samples were dried in a vacuum oven at 45°C, milled to a fine powder and then stored at 4°C for further analysis. Chemical composition analysis The moisture content of the samples was determined according to AOAC method 930.15 41 . Ash content was determined according to AOAC method 942.05 41 . Crude protein content was determined using the Dumas combustion method in a Vario MAX Cube CN analyser (Elementar, Stockport, UK). A conversion factor of 6.25 was used to convert total nitrogen to crude protein content. Crude fibre content was determined according to the AOCS method Ba 6a-05 42 in an ANKOM A2000 fibre analyser (ANKOM Technology, NY, US). Free sugars (glucose, fructose and sucrose) were determined by mixing 200 mg of sample with 1 mL of 10% ethanol solution. Sugars were extracted by shaking at room temperature (25°C) for 1 h, followed by centrifugation at 21000 x g for 10 min with the supernatants stored at 4°C and the pellets discarded. The supernatants containing free sugars were filtered through a 0.22 \({\mu }\) m membrane and quantified by HPLC in a REZEX-ROA Organic acid H+ (8%) Ion exclusion column with a refractive index (RI) detector. Total starch content was determined according to the AOAC method 996.11 41 using an amyloglucosidase/ \({\alpha }\) -amylase assay kit (Megazyme, Wicklow, Ireland). The supernatants containing hydrolysed starch were filtered through a 0.22 \({\mu }\) m membrane and quantified by HPLC in a REZEX-ROA Organic acid H+ (8%) Ion exclusion column with a refractive index (RI) detector. Measurements were done once per experimental replicate. All values are reported as percent dry matter basis (% DM). Amino acid profile analysis Total AAs were prepared by mixing 200 mg of milled, dry SSF samples with 5 mL of 6 M HCl (0.1% phenol) in 15 mL glass tubes, degassed and sealed under an N 2 stream, and incubated for 24 h at 110°C and allowed to cool to room temperature. The hydrolysates were adjusted with 6 M NaOH to pH 6–8, made up to a final volume of 50 mL with water, and then centrifuged at 21000 x g for 10 min, with the supernatant filtered through a 0.22 \({\mu }\) m membrane and added in a 1:1 ratio to a 250 pmol/ \({\mu }\) L solution of DL-norvaline as an internal standard. Pre-column derivatisation of amino acids with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was performed in accordance with Reverter, et al. 43 . Briefly, 70 \({\mu }\) L of borate buffer was added to 10 \({\mu }\) L of AA hydrolysate, or amino acid standards, and 20 \({\mu }\) L of AQC solution (4 mg AQC in 1 mL anhydrous acetonitrile). After 1 min incubation at room temperature, the contents were transferred to an autosampler vial and capped with a silicone-lined septum. Derivatised samples were quantified by HPLC using an AccQ-Tag Amino acids C18 column with variable wavelength (VWD) (260 nm detection) and fluorescence (FLD) (266 nm emission and 473 nm detection) detectors. Mobile phases were composed of water (solvent A), acetonitrile (solvent B) and 50 mM ammonium formate (adjusted to pH 2.9 ± 0.05 with formic acid) (solvent C). Elution was carried out as follows: 0-0.5 min 0–1% solvent B and 100 − 99% solvent C, 0.5–15 min 1–4% solvent B and 99 − 96% solvent C, 15–23 min 4–5% solvent B and 96 − 95% solvent C, 23–24 min 5–9% solvent B and 95 − 91% solvent C, 24–28 min 9–14% solvent B and 91 − 86% solvent C, 28–45 min 14–17% solvent B and 86 − 83% solvent C, 45–55 min 17–60% solvent B and 83 − 0% solvent C. The flow rate was 1 mL/min, and the injection volume was 10 \({\mu }\) L. The AA in the samples were analysed according to retention time and peak area. Statistical analysis The analytical data was analysed by two-tailed analysis of variance (ANOVA) followed by Tukey’s HSD post-hoc test using IBM SPSS v 28 statistical software (IBM UK Ltd, Hampshire, UK). The effect of the experimental treatments was analysed with a one-way ANOVA by comparing the results from each measurement at each time point (0, 24, 48 and 72 h) in two groups, BC/W and BC/GJ, and the different levels of BC/GJ/GS. The effect of time in the fermentation and the interaction between the experimental treatment and fermentation time, was analysed by a two-way ANOVA in two separate groups, BC/W and BC/GJ (supplementary Table S1 ), and the different levels of BC/GJ/GS (supplemental Table S2 ). Lastly, all experimental treatments at 72 h of SSF and those of unfermented BC were compared to identify the significant, most impactful changes with a one-way ANOVA followed by Tukey’s HSD post-hoc test (supplementary Table S3 ). The results were expressed as mean value \(\pm\) standard error. A statistical significance level ( \({\alpha }\) 0.05) was used to analyse the results. Declarations Data availability The data supporting the findings reported herein are available on request from the corresponding author. Acknowledgements The authors would like to thank the BBSRC FoodBiosystems DTP committee and the staff of IBERS, Aberystwyth University for their support. This research was funded by the BBSRC, UKRI doctoral training grant no: BB/T008776/1. The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Author information Authors and Affiliations Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Aberystwyth, SY23 3EE, UK Juan Felipe Sandoval, Joe Gallagher & David N. Bryant * Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Faculty of Pharmacy, Universitat de València, Avda. Vicent Andrés Estellés, s/n, 46100 Burjassot, València, Spain Julia Rodriguez-Garcia Samworth Brothers Limited, Leicestershire, LE13 1GA, UK Kerry Whiteside * Corresponding author. Tel: +44 07432487541. E-mail: [email protected] Contributions All authors designed this study, reviewed and approved the manuscript. 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Substrate BC/W BC/GJ Composition 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h EAA Histidine 2.08 ± 0.1 a 1.76 ± 0.1 a 1.87 ± 0.1 a 1.90 ± 0.1 a 1.61 ± 0.2 a 1.61 ± 0.1 a 1.85 ± 0.1 a 1.98 ± 0.2 a Isoleucine 2.47 ± 0.1 a 2.72 ± 0.2 a 3.00 ± 0.1 a 2.78 ± 0.0 a 2.28 ± 0.4 b 3.25 ± 0.2 a 2.85 ± 0.3 a 2.68 ± 0.3 b Leucine 5.00 ± 0.1 a 4.62 ± 0.4 a 4.74 ± 0.2 a 4.42 ± 0.1 a 4.58 ± 0.5 a 5.04 ± 0.4 a 4.17 ± 0.4 a 4.50 ± 0.5 a Lysine 2.94 ± 1.2 a 4.74 ± 1.8 a 4.62 ± 1.6 a 4.54 ± 0.4 a 1.18 ± 0.1 a 1.77 ± 0.1 a 3.39 ± 0.2 a 3.17 ± 0.5 a Methionine 1.30 ± 0.0 a 1.39 ± 0.3 a 1.25 ± 0.2 a 0.81 ± 0.1 a 1.02 ± 0.3 b 0.65 ± 0.0 a 0.70 ± 0.1 b 1.19 ± 0.3 a Phenylalanine 3.18 ± 0.1 a 2.93 ± 0.2 a 2.80 ± 0.1 a 2.44 ± 0.1 a 3.55 ± 0.4 a 3.63 ± 0.2 a 2.94 ± 0.3 a 3.38 ± 0.2 b Threonine 2.18 ± 0.1 a 2.42 ± 0.2 a 2.69 ± 0.1 a 2.60 ± 0.0 a 2.00 ± 0.2 a 2.57 ± 0.2 a 2.78 ± 0.2 a 3.05 ± 0.2 b Valine 4.28 ± 0.2 a 4.87 ± 0.8 a 5.35 ± 0.4 a 4.97 ± 0.1 a 3.03 ± 0.4 b 3.70 ± 0.2 a 3.33 ± 0.3 b 3.59 ± 0.3 b NEAA Alanine 2.71 ± 0.1 a 3.82 ± 0.4 a 4.94 ± 0.2 a 5.77 ± 0.3 a 2.28 ± 0.2 a 3.86 ± 0.3 a 4.12 ± 0.3 a 4.62 ± 0.5 b Aspartic acid 3.18 ± 0.1 a 5.02 ± 0.4 a 6.81 ± 0.3 a 6.08 ± 0.2 a 3.55 ± 0.3 a 5.17 ± 0.2 a 5.91 ± 0.4 a 7.17 ± 0.9 a Cysteine 0.28 ± 0.0 a 0.18 ± 0.0 a 0.26 ± 0.0 a 0.30 ± 0.1 a 0.30 ± 0.1 b 0.18 ± 0.0 a 0.16 ± 0.0 a 0.37 ± 0.1 a Glutamic acid 25.58 ± 0.9 a 18.68 ± 1.4 a 17.20 ± 0.7 a 13.45 ± 0.5 a 27.03 ± 3.0 a 19.99 ± 2.3 a 12.28 ± 1.1 b 15.35 ± 2.7 a Glycine 1.97 ± 0.1 a 2.04 ± 0.2 a 2.37 ± 0.2 a 2.18 ± 0.0 a 2.85 ± 0.4 b 3.76 ± 0.2 b 3.83 ± 0.2 b 3.76 ± 0.4 b Proline 8.72 ± 0.2 a 7.02 ± 0.6 a 6.76 ± 0.2 a 5.40 ± 0.2 a 7.87 ± 0.8 a 6.94 ± 0.8 a 4.27 ± 0.5 b 5.17 ± 0.7 b Serine 3.19 ± 0.1 a 2.79 ± 0.2 a 2.82 ± 0.1 a 2.65 ± 0.1 a 3.26 ± 0.4 a 3.18 ± 0.3 a 2.89 ± 0.2 a 3.56 ± 0.4 b Tyrosine 3.73 ± 0.5 a 4.30 ± 1.0 a 4.00 ± 0.7 a 3.62 ± 0.1 a 1.64 ± 0.6 b 1.34 ± 0.1 a 1.64 ± 0.1 b 2.61 ± 0.5 b Arginine 3.70 ± 0.2 a 3.38 ± 0.2 a 3.84 ± 0.2 a 3.76 ± 0.1 a 2.56 ± 0.3 b 2.86 ± 0.2 a 3.42 ± 0.2 a 3.78 ± 0.3 a Sum AA 76.49 ± 2.2 a 72.66 ± 8.3 a 75.32 ± 5.2 a 67.65 ± 0.7 a 70.59 ± 7.6 a 69.51 ± 5.8 a 60.55 ± 4.7 a 69.93 ± 7.5 a Sum EAA 23.43 ± 1.2 a 25.45 ± 4.0 a 26.33 ± 2.7 a 24.45 ± 0.1 a 19.26 ± 2.3 a 22.22 ± 1.4 a 22.03 ± 1.8 a 23.54 ± 1.9 a Sum NEAA 53.06 ± 1.8 a 47.21 ± 4.4 a 48.99 ± 2.5 a 43.20 ± 0.7 a 51.34 ± 5.5 a 47.29 ± 4.4 a 38.52 ± 3.0 a 46.39 ± 5.8 a % EAA/AA 30.63 ± 1.5 a 34.63 ± 0.7 a 34.80 ± 0.4 a 36.15 ± 1.5 a 27.07 ± 1.3 a 32.03 ± 1.9 a 36.38 ± 1.2 a 34.00 ± 0.5 a % NEAA/AA 69.37 ± 1.5 a 65.37 ± 0.7 a 65.20 ± 0.4 a 63.85 ± 1.5 a 72.93 ± 1.3 a 67.97 ± 1.9 a 63.62 ± 1.2 a 66.00 ± 0.5 a CP: Crude protein, AA: Amino acids EAA: Essential amino acids, NEAA: Non-essential amino acids Table 2 . Total amino acid profile [g/100g CP] from bread crusts (BC)/green juice (GJ)/dry green solids (GS) to CP 19%, 20%, 23%, 27% and 29% DM at 0 and 72 h of solid-state fermentation (SSF). Results are expressed as the mean value ± a standard error. Data with different letters in the same time point are significantly different (p < 0.05) between the different levels of GS, following Tukey´s HSD post-hoc test. Data with the same letters are statistically similar. CP 0 h [%DM] 19% 20% 23% 27% 29% Composition 0 h 72 h 0 h 72 h 0 h 72 h 0 h 72 h 0 h 72 h EAA Histidine 1.18 ± 0.2 a 1.36 ± 0.2 a 1.87 ± 0.2 a 2.47 ± 0.0 ab 2.40 ± 0.1 a 5.18 ± 0.1 c 3.37 ± 1.1 a 2.72 ± 0.0 b 6.55 ± 0.2 b 4.17 ± 0.4 c Isoleucine 1.57 ± 0.2 a 1.86 ± 0.0 a 2.53 ± 0.0 b 2.19 ± 0.1 a 2.94 ± 0.1 b 2.45 ± 0.1 a 2.81 ± 0.1 b 2.60 ± 0.0 a 3.70 ± 0.1 c 3.62 ± 0.3 b Leucine 4.13 ± 0.6 a 3.85 ± 0.0 a 6.03 ± 0.2 bc 4.49 ± 0.1 ab 6.47 ± 0.1 bc 4.91 ± 0.2 ab 5.94 ± 0.1 b 5.39 ± 0.0 b 7.74 ± 0.3 c 7.01 ± 0.4 c Lysine 1.56 ± 0.3 a 1.90 ± 0.0 a 2.24 ± 0.0 ab 2.36 ± 0.2 ab 2.77 ± 0.1 b 1.42 ± 0.1 a 2.41 ± 0.1 b 2.17 ± 0.0 a 2.22 ± 0.1 ab 3.94 ± 0.6 b Methionine 1.74 ± 0.1 a 1.53 ± 0.0 a 2.09 ± 0.0 a 1.50 ± 0.1 a 2.24 ± 0.1 a 2.03 ± 0.0 ab 2.31 ± 0.2 a 2.00 ± 0.3 ab 2.28 ± 0.1 a 2.23 ± 0.0 b Phenylalanine 3.21 ± 0.4 a 3.04 ± 0.2 a 4.89 ± 0.3 a 3.32 ± 0.2 a 5.38 ± 0.2 a 7.62 ± 0.2 b 6.00 ± 0.8 a 6.56 ± 0.6 b 9.92 ± 0.5 b 6.67 ± 0.7 b Threonine 1.95 ± 0.3 a 2.59 ± 0.1 a 3.03 ± 0.2 ab 2.75 ± 0.1 a 3.49 ± 0.2 b 4.31 ± 0.2 b 3.53 ± 0.2 b 3.77 ± 0.1 b 5.33 ± 0.1 c 4.51 ± 0.2 b Valine 3.80 ± 0.7 a 2.82 ± 0.0 a 3.89 ± 0.1 a 2.97 ± 0.1 a 4.48 ± 0.1 a 3.09 ± 0.0 ab 4.53 ± 0.4 a 3.79 ± 0.7 ab 4.55 ± 0.2 a 4.78 ± 0.0 b NEAA Alanine 2.54 ± 0.5 a 3.93 ± 0.1 a 3.85 ± 0.1 ab 4.45 ± 0.1 ab 4.28 ± 0.1 b 3.77 ± 0.3 a 4.15 ± 0.2 b 3.86 ± 0.1 a 5.07 ± 0.0 b 5.84 ± 0.7 b Aspartic acid 4.52 ± 1.0 a 6.76 ± 0.8 a 6.62 ± 0.2 a 7.12 ± 0.0 ab 7.59 ± 0.2 a 5.46 ± 0.4 a 6.65 ± 0.8 a 5.00 ± 0.3 a 6.93 ± 0.1 a 10.06 ± 0.8 b Cysteine 0.61 ± 0.2 a 0.43 ± 0.0 a 0.64 ± 0.0 a 0.42 ± 0.0 a 0.44 ± 0.0 a 0.64 ± 0.0 a 0.52 ± 0.1 a 0.44 ± 0.1 a 0.58 ± 0.0 a 0.57 ± 0.1 a Glutamic acid 23.14 ± 3.0 bc 11.49 ± 1.1 ab 25.08 ± 1.8 c 11.10 ± 0.2 ab 20.44 ± 0.5 abc 8.92 ± 0.5 a 13.05 ± 2.0 ab 7.36 ± 0.5 a 11.81 ± 0.7 a 13.17 ± 1.0 b Glycine 2.22 ± 0.4 a 2.42 ± 0.0 a 3.37 ± 0.0 b 2.87 ± 0.3 a 3.59 ± 0.0 b 8.36 ± 4.5 a 3.45 ± 0.2 b 3.76 ± 0.1 a 5.09 ± 0.1 c 4.27 ± 0.3 a Proline 6.27 ± 0.8 a 3.77 ± 0.1 a 7.52 ± 0.1 a 3.90 ± 0.2 a 6.97 ± 0.1 a 4.67 ± 0.0 a 5.59 ± 0.1 a 4.06 ± 0.1 a 5.70 ± 0.2 a 6.24 ± 1.3 a Serine 2.91 ± 0.4 a 3.03 ± 0.1 a 4.06 ± 0.2 ab 3.07 ± 0.1 a 4.27 ± 0.1 bc 4.55 ± 0.2 b 3.87 ± 0.1 ab 3.86 ± 0.0 b 5.45 ± 0.0 c 4.51 ± 0.2 b Tyrosine 3.37 ± 0.1 a 2.94 ± 0.1 a 3.99 ± 0.1 a 2.91 ± 0.1 a 4.53 ± 0.4 a 4.52 ± 0.1 a 5.17 ± 0.7 a 5.24 ± 1.4 a 5.18 ± 0.3 a 5.24 ± 0.1 a Arginine 2.29 ± 0.3 a 3.30 ± 0.4 a 4.20 ± 0.5 ab 4.11 ± 0.2 a 5.24 ± 0.4 ab 8.34 ± 0.3 c 6.15 ± 1.0 b 6.70 ± 0.1 b 10.97 ± 0.0 c 7.60 ± 0.3 bc Sum AA 67.03 ± 7.2 a 57.02 ± 0.6 a 85.89 ± 0.4 ab 62.01 ± 1.7 ab 87.51 ± 1.7 b 80.22 ± 2.6 bc 79.50 ± 2.4 ab 69.28 ± 2.3 ab 99.05 ± 0.7 b 94.42 ± 6.4 c Sum EAA 19.15 ± 1.1 a 18.95 ± 0.6 a 26.57 ± 0.8 ab 22.05 ± 0.5 a 30.16 ± 1.0 b 31.00 ± 0.7 bc 30.90 ± 2.8 b 29.00 ± 1.6 b 42.29 ± 0.3 c 36.92 ± 1.8 c Sum NEAA 47.88 ± 6.1 38.07 ± 1.3 a 59.31 ± 1.2 39.96 ± 1.3 a 57.35 ± 0.7 49.22 ± 3.3 ab 48.60 ± 0.4 40.28 ± 0.6 a 56.77 ± 0.4 57.50 ± 4.6 c % EAA/AA 28.72 ± 1.4 a 33.24 ± 1.5 a 30.94 ± 1.1 a 35.56 ± 0.3 ab 34.45 ± 0.5 ab 38.71 ± 2.1 ab 38.80 ± 2.4 bc 41.83 ± 1.0 b 42.69 ± 0.0 c 39.15 ± 0.7 ab % NEAA/AA 71.28 ± 1.4 c 66.76 ± 1.5 b 69.06 ± 1.1 c 64.44 ± 0.3 ab 65.55 ± 0.5 bc 61.29 ± 2.1 ab 61.20 ± 2.4 ab 58.17 ± 1.0 a 57.31 ± 0.0 a 60.85 ± 0.7 ab CP: Crude protein, DM: Dry matter, AA: Amino acids EAA: Essential amino acids, NEAA: Non-essential amino acids Table 3 . Essential amino acid (EAA) reference ratios calculated against the FAO nutritional guidelines (g/100g CP) for adults for unfermented bread crusts (BC), and solid-state fermentation (SSF) substrates BC/water (W), BC/green juice (GJ), BC/GJ/green solids (GS) to CP 19%, 20%, 23%, 27% and 29% DM at 72 h. EAA FAO requirement BC Substrate after 72 h SSF BC/W BC/GJ BC/GJ/GS to % DM CP 19% 20% 23% 27% 29% Histidine 1.5 1.38 1.27 1.32 0.91 1.65 3.45 1.82 2.78 Isoleucine 3 0.82 0.93 0.89 0.62 0.73 0.82 0.87 1.21 Leucine 5.9 0.85 0.75 0.76 0.65 0.76 0.83 0.91 1.19 Lysine 4.5 0.65 1.01 0.70 0.42 0.52 0.32 0.48 0.88 Threonine 2.3 0.95 1.13 1.33 1.13 1.19 1.87 1.64 1.96 Valine 3 1.43 1.66 1.20 0.94 0.99 1.03 1.26 1.59 Methionine + Cysteine 2.2 0.72 0.50 0.71 0.89 0.87 1.21 1.11 1.27 Phenylalanine + Tyrosine 3 2.30 2.02 2.00 1.99 2.08 4.04 3.93 3.97 Tryptophan 0.6 CP: Crude protein, DM: Dry matter Additional Declarations (Not answered) Supplementary Files Paper1SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 16 Nov, 2024 Read the published version in npj Science of Food → Version 1 posted Editorial decision: revise 24 Jul, 2024 Review # 3 received at journal 13 Jul, 2024 Reviewer # 3 agreed at journal 01 Jul, 2024 Reviewer # 2 agreed at journal 18 Jun, 2024 Review # 1 received at journal 21 May, 2024 Reviewer # 1 agreed at journal 03 May, 2024 Reviewers invited by journal 12 Mar, 2024 Submission checks completed at journal 21 Feb, 2024 First submitted to journal 20 Feb, 2024 Editor assigned by journal 20 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3973183","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":278568081,"identity":"3fe7f1aa-7dfd-4ab3-a5cb-7876676f00fb","order_by":0,"name":"David Bryant","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYDCCGwyMDxgbYLwDxGlhNiBZC5sEaVr4brdfq/i5wyafgf3wA2aeM0Rokbxzpuxm75k0ywaeNANmnhtEaDG4kZN2g7ftsAEDQw4DM88HIrUU/m37b8DA/4ZoLenHmHnbDhgwSIBsIcZhQL8wS8ueSTZgk3hmcHAOMd4HhtjDj2932Bnw8yc/fPDmGBFaGBh4DMAUGwOREQkE7A+IVDgKRsEoGAUjFgAAcg052JtopRIAAAAASUVORK5CYII=","orcid":"","institution":"Aberystwyth University","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Bryant","suffix":""},{"id":278568082,"identity":"0607faf7-fdc9-4fd5-982a-c28f6310c9fa","order_by":1,"name":"Juan Sandoval","email":"","orcid":"https://orcid.org/0000-0002-2307-3873","institution":"Aberystwyth University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Sandoval","suffix":""},{"id":278568083,"identity":"f19f59cd-c589-4534-a5b7-a4ac7c38c831","order_by":2,"name":"Joe Gallagher","email":"","orcid":"","institution":"Aberystwyth University","correspondingAuthor":false,"prefix":"","firstName":"Joe","middleName":"","lastName":"Gallagher","suffix":""},{"id":278568084,"identity":"40d4e61b-de15-417c-8584-384d1073bf37","order_by":3,"name":"Julia Rodriguez-Garcia","email":"","orcid":"https://orcid.org/0000-0002-4986-3144","institution":"Universitat de València","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Rodriguez-Garcia","suffix":""},{"id":278568085,"identity":"88b8fc7c-b90e-48c5-abe7-d2a20bef9ac5","order_by":4,"name":"Kerry Whiteside","email":"","orcid":"","institution":"Samworth Brothers Ltd","correspondingAuthor":false,"prefix":"","firstName":"Kerry","middleName":"","lastName":"Whiteside","suffix":""}],"badges":[],"createdAt":"2024-02-20 15:27:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3973183/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3973183/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41538-024-00338-y","type":"published","date":"2024-11-16T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52661899,"identity":"ba617cbf-6c45-4f56-8b28-cdbcfca11dc5","added_by":"auto","created_at":"2024-03-14 08:05:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":646099,"visible":true,"origin":"","legend":"\u003cp\u003e(I) Photographic record of SSF experiments. (a) bread crusts (BC)/water (W), (b) BC/green juice (GJ), (c) BC/GJ/dry green solids (GS) to CP 19% DM, (d) BC/GJ/GS to CP 20% DM, (e) BC/GJ/GS to CP 23% DM, (f) BC/GJ/GS to CP 27% DM and (g) BC/GJ/GS to CP 29% DM at 0, 24, 48 and 72 h of fermentation. (II) % of mass loss of substrate at 24, 48 and 72 h of fermentation. (a) BC/GJ and BC/W and (b) BC/GJ/GS to CP 19%, 20%, 23%, 27% or 29% DM. Bars represent the mean value and vertical lines a standard error. Data with different letters in the same time point are significantly different (p \u0026lt; 0.05), following Tukey´s HSD post-hoc test. Data without letters are statistically similar.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-3973183/v1/e47cc693b08de23c2f439fcc.png"},{"id":52661897,"identity":"1fd01650-eccc-423e-8937-a261b503c001","added_by":"auto","created_at":"2024-03-14 08:05:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":132127,"visible":true,"origin":"","legend":"\u003cp\u003eChemical composition analysis. (a) Bread crusts (BC)/water (W) and BC/green juice (GJ) at 0, 24, 48 and 72 h of solid-state fermentation (SSF). (b) BC/GJ/dry green solids (GS) to CP 19%, 20%, 23%, 27% and 29% DM at 0 and 72 h of solid-state fermentation (SSF). Bars represent the mean value and vertical lines a standard error. Data with different letters in the same time point are significantly different (p \u0026lt; 0.05), following Tukey´s HSD post-hoc test. Data without letters are statistically similar.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3973183/v1/e52e06df3371201042679ab9.png"},{"id":52661896,"identity":"47feb5c6-1a62-4938-ab38-78ec5e74779a","added_by":"auto","created_at":"2024-03-14 08:05:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42891,"visible":true,"origin":"","legend":"\u003cp\u003eRatio of crude protein to starch. (a) Bread crusts (BC)/water (W) and BC/green juice (GJ) at 0, 24, 48 and 72 h of solid-state fermentation (SSF). (b) BC/GJ/dry green solids (GS) to CP 19%, 20%, 23%, 27% and 29% DM at 0 and 72 h of solid-state fermentation (SSF). Bars represent the mean value and vertical lines a standard error. Data with different letters in the same time point are significantly different (p \u0026lt; 0.05), following Tukey´s HSD post-hoc test. Data without letters are statistically similar.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-3973183/v1/685700f6ccd59b9c2ca03371.png"},{"id":69171925,"identity":"9c48f2fa-d438-4672-8bca-c84ae47ca4c5","added_by":"auto","created_at":"2024-11-17 08:05:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2125395,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3973183/v1/d97b3c99-85f4-49ec-8d57-9f1cfbd41741.pdf"},{"id":52661898,"identity":"7bd6cbe3-f893-4aaa-a4e9-d352814a012a","added_by":"auto","created_at":"2024-03-14 08:05:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2069251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Paper1SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3973183/v1/afa06e12d8ef0da168662c87.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Improved Nutritional Value of Surplus Bread and Perennial Ryegrass Extracts Via Solid-State Fermentation (SSF) with Rhizopus oligosporus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) is one of the most consumed crops worldwide, contributing to 20% of the calories and proteins in human diets\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and is the basis of multiple staple foods across different cultures, such as breads, cakes, biscuits, cookies and crackers. Approximately 760\u0026nbsp;million tons of wheat flour are produced yearly worldwide\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, of which 10% is wasted\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Bakery waste, particularly in the form of bread, is a growing global concern, as 270\u0026nbsp;million tons of bakery products were produced in 2015, of which 69\u0026nbsp;million tons were wasted. Some of this waste occurs at the household level, mainly due to spoilage, but also at the manufacture and retail level, where bread waste is called surplus and is primarily comprised of bread crusts and breadcrumbs. While most of this surplus is safe for consumption, it is unsuitable for commercialisation, as it does not comply with the quality specifications given by the manufacturer to the relevant legal authorities, such as the UK Food Standards Agency (FSA), regarding physicochemical composition, and stablished by the manufacturer for appearance and sensory characteristics.\u003c/p\u003e \u003cp\u003eThe valorisation of surplus bread has been widely explored. Traditionally, it is either milled to be reincorporated into the production of new batches of bread or other products such as soups and fried foods\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e or as an animal feed\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, but due to the volume of waste generated not all of it is given proper use and ends up being discarded. Ben Rejeb, et al. \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e reviewed the available routes for the valorisation of bread waste into value-added products, both through chemical processes and fermentation, including the manufacturing of ethanol, lactic acid, succinic acid, biohydrogen, hydroxymethylfurfural, isolated proteins, pigments, sugar syrups, aromatic compounds, and enzymes. More recently, solid-state fermentation (SSF) has also been explored as an alternative valorisation process, as bread possesses most of the nutrients needed by SSF microorganisms, specifically filamentous fungi\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, and provides a structural matrix of gluten, the main protein of wheat, and gelatinised starch in which they can grow\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSSF has been used to nutritionally enhance the biomass of cereals, legumes, fruits and vegetables for human consumption\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, with a low environmental impact. The lack of a liquid phase reduces the generated wastewater and other potential pollutants\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, while the use of waste materials as substrates minimises the cost of media preparation and reduces the energy consumption of the process, as sterilisation is not commonly required\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Amongst the multiple filamentous fungi reported in SSF in the food industry, \u003cem\u003eRhizopus microsporus\u003c/em\u003e var. \u003cem\u003eoligosporus (Rmo)\u003c/em\u003e has been used for centuries in the production of tempeh from soybeans (\u003cem\u003eGlycine max\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eRmo\u003c/em\u003e has been shown to valorise surplus bread, reducing the concentration of starch (from 65.8\u0026ndash;42.7%) while increasing the concentration of crude protein (from 13.2\u0026ndash;16.1%) and crude fibres (from 11.6\u0026ndash;26.7%)\u003csup\u003e12\u003c/sup\u003e and to increase the relative ratio of essential amino acids (AA), minerals and vitamins\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eRmo\u003c/em\u003e, like other filamentous fungi, can produce proteases that cleave protein, thereby altering the AA profile and increasing the concentration of essential AA\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, improving the protein quality. However, as \u003cem\u003eRmo\u003c/em\u003e is incapable of nitrogen fixation, increasing the total nitrogen content in the substrate requires external supplementation.\u003c/p\u003e \u003cp\u003eAlternative protein sources with high unexploited potential for human applications are forage grasses and clovers. These crops have been studied as a source of fibres and cellulose\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, fertilisers\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, biogas and bioethanol\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and lactic acid\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e as well as for direct human consumption\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, with its primary use being animal feed. These crops are rich in high quality protein, and the mineral, i.e., magnesium, potassium, phosphorus and calcium\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and nutraceutical content, i.e., fructan, pinitol, isoflavones and tannins\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, also contribute to their nutritional profile. They can achieve high biomass yields and protein outputs; for instance a square hectare of land can produce between 1.8 and 3 tons of alfalfa protein while the same landmass only 150 to 200 kg of meat protein\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, with low environmental footprints\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, which makes them widely grown worldwide.\u003c/p\u003e \u003cp\u003eOf these crops, PRG (\u003cem\u003eLolium perenne)\u003c/em\u003e is one of the most used grass species in the world, especially in Europe where it represents up to 50% of the grass seed market\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. It\u0026rsquo;s mainly used as feed in grazing livestock for animal protein production, which compared to using its protein for direct food applications, has been reported to be an inefficient use of the land\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. It contains a crude protein (CP) content of approximately 12% (dry matter (DM)) and a similarly well-balanced amino acid profile as soybean\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, due to the composition of RuBisCO (ribulose-1,5-bisphosphate carboxylase-oxygenase), a key enzyme in the fixation of CO\u003csub\u003e2\u003c/sub\u003e during photosynthesis, which makes up to 50% of the total soluble protein content\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Despite this, PRG is not adequate for direct human consumption due to its high content of undigestible fibres, particularly cellulose, which can range between 30% and 50% DM\u003csup\u003e29\u003c/sup\u003e, and its unpalatability. Extraction, fractionation, purification and/or chemical and biological conversion of the protein in forage crops is required to make use of it in food applications and has been widely explored by different methods, although with an emphasis on animal feed\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThus, this research project proposes a novel sustainable process to valorise surplus bread crusts and PRG biomass, a combination of substrates reported here for the first time in the literature. It is hypothesized that the protein content and the concentration of available essential amino acids in the final product will increase by combining these substrates via SSF with \u003cem\u003eRmo\u003c/em\u003e. This work provides a fundamental study for the application of forage crop proteins in producing an alternative source of high-quality protein for human consumption via SSF, which would provide useful information for future developments in novel foods.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eSolid-State fermentation\u003c/p\u003e\n\u003cp\u003eSurplus bread crusts (BC) are a relatively dry material (79% DM) with a CP content of 16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2% DM, crude fibre content of 15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2% DM and starch content of 69.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% DM that can be transformed into an alternative food via SSF as part of a strategy in the management of food waste. The porosity of the structure makes the nutrients accessible for \u003cem\u003eRmo\u003c/em\u003e, which allows the mycelia to grow throughout the whole substrate and not just superficially, as has been seen in soybeans where the mycelium can penetrate approximately 2 mm into the substrate within 40 h of fermentation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Exploiting the structure and dimensions of BC was fundamental in accelerating the growth of \u003cem\u003eRmo.\u003c/em\u003e Firstly, the use of 1x1x1 cm BC cubes allowed the fungi to grow throughout the substrate (as sown in \u003cstrong\u003eFigure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e), in contrast to SSF with milled BC (as shown in \u003cstrong\u003eFigure S2\u003c/strong\u003e) which only supported superficial growth as \u003cem\u003eRmo\u003c/em\u003e could not penetrate the substrate. Secondly, the initial substrate moisture of 56% was optimised from a range of 40 to 70% to the maximum consumption of starch after 72 h of fermentation (as shown in \u003cstrong\u003eFigure S3\u003c/strong\u003e). Thirdly, a pH of 3.5 was selected to avoid the growth of undesirable microorganisms, which was proven to not significantly affect the growth of \u003cem\u003eRmo\u003c/em\u003e (as shown in \u003cstrong\u003eFigure S4\u003c/strong\u003e). This adjustment was significant due to the addition of grass juice (GJ), a fresh screw-pressed juice of PRG which contained microorganisms that could influence the process. GJ and dry green solids (GS) were added to increase the nutritional value of the SSF substrate in an environmentally sustainable way, as these materials had CP contents of 16.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7% DM and 34.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% DM, respectively. The first section of experiments, comparing BC/W and BC/GJ, were conducted to provide a baseline of the SSF process and understand the influence of the plant matrix on \u003cem\u003eRmo\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the second section, which compared increasing amounts of GS to increase the CP content, were performed to evaluate alterations in the amino acid profile of the protein after the fermentation (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe SSF experimental designs were photographed every 24 h from start to end of the fermentation to record the growth of \u003cem\u003eRmo\u003c/em\u003e over the different BC substrates (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Increasing amounts of PRG protein were added to BC (19%, 20%, 23%, 27% or 29% DM) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI, c-g), and this consistently slowed down the growth of \u003cem\u003eRmo\u003c/em\u003e mycelium. After 24 h of SSF and up to 20% DM CP BC/GJ/GS (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI, d), the fungi was able to superficially cover the whole dish. However, higher quantities of GS (BC/GJ/GS to CP 23%, 27% or 29% DM) reduced this growth (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI, e, f and g, respectively). This deceleration could possibly be a consequence of phenolic compounds in PRG, which, while not as widely studied as in other crops such as red and white clover, have shown to contain organic acids such as gallic acid and protocatechuic acid and flavonoids such as quercetin and kaempferol\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, all of which have been proven to have antifungal activity at concentrations over 0.3 mg/mL\u003csup\u003e32\u003c/sup\u003e. These concentrations are considerably higher than those naturally found in whole PRG and even less so in GJ, which could explain why inhibition of growth was only evident in higher concentrations of GS. After 48 h, \u003cem\u003eRmo\u003c/em\u003e mycelium had completely covered all samples up to BC/GJ/GS to CP 27% DM (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI, a-f), with BC/GJ/GS to CP 29% DM (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI, g) being partially covered, and after 72 h the fungi started to senesce as noted by the change in colour from white-light grey to yellow-dark grey. This timeframe aligns with the ageing of \u003cem\u003eRmo\u003c/em\u003e in SSF and implies the start of the deterioration of the mycelium, the degradation of the nutritional value and negative changes to the sensory profile of the substrate\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e (II) details the percentage loss of total substrate mass at 24, 48 and 72 h versus the initial mass at 0 h as an approximate quantitative measure of the growth of the fungi during SSF. A fraction of the mass lost was due to the evaporation of water, while the remainder was attributed to carbohydrate metabolism of the fungi and the production of CO\u003csub\u003e2\u003c/sub\u003e, although the extent by which each contributes to the mass change was not measured. A mathematical model developed by Figueroa-Montero, et al. \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e in a SFF with \u003cem\u003eAspergillus niger\u003c/em\u003e shows the complexity of the mass transfer phenomena in SSF systems, where water and CO\u003csub\u003e2\u003c/sub\u003e are the main outputs of the fermentation. The mass loss of BC/GJ and BC/W (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e II, a) was statistically different across time (p\u0026thinsp;=\u0026thinsp;0.00) and between experimental treatments (p\u0026thinsp;=\u0026thinsp;0.00) at 24 and 72 h, although no interaction was found between the factors (p\u0026thinsp;=\u0026thinsp;0.16) (\u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e), with BC/W having a 49.4% higher loss at 24 h and 14.0% higher loss at 72 h; however, no significant differences in mass loss were observed at 48 h (p\u0026thinsp;=\u0026thinsp;0.08). The mass loss of BC/GJ at different levels of GS (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e II, b) had statistical differences across time (p\u0026thinsp;=\u0026thinsp;0.00) and the different experimental treatments (p\u0026thinsp;=\u0026thinsp;0.00), but no interaction effect was identified (p\u0026thinsp;=\u0026thinsp;0.19) (\u003cstrong\u003eTable S2\u003c/strong\u003e), with the highest losses generally occurring at the treatments with 19% and 29% DM and the lowest losses with 23% and 27% DM.\u003c/p\u003e\n\u003cp\u003eChemical composition analysis\u003c/p\u003e\n\u003cp\u003eThe chemical composition of the substrates was assessed to evaluate the change in nutritional properties over the course of the SSF (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The DM content of all experimental units started at 45.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4% and ended at 43.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%, without substantial differences at any time point. Although the ambient moisture in the SSF chamber was not controlled, the DM was stable throughout the process. The main driver for the mass change in all macronutrients was the digestion of starch as the primary carbon source of the substrate. These changes have also been described in the literature, as fermentation with \u003cem\u003eRmo\u003c/em\u003e transforms carbohydrates into fungal biomass, thereby increasing the concentration of crude protein and crude fibre\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Additionally, the ratio of crude protein to starch content was calculated to help visualise the changes in the chemical composition in terms of the nutritional value of the substrate (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eComparing the composition of BC/GJ and BC/W (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea), the nutritional value of BC improved considerably in both cases, and few differences were identified. Crude protein content was equal between the experimental treatments (p\u0026thinsp;=\u0026thinsp;0.66) at each time point, but it significantly increased (p\u0026thinsp;=\u0026thinsp;0.00) over time, and no significant interaction was observed between factors (p\u0026thinsp;=\u0026thinsp;0.38) (\u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). Analysing the change of both treatments over time (\u003cstrong\u003eFigure S5\u003c/strong\u003e) shows an increase in the crude protein content of 74.6% after 72 h of SSF. Crude fibre content was significantly higher (p\u0026thinsp;=\u0026thinsp;0.01) for BC/GJ at 72 h, likely due to the faster carbohydrate metabolism in BC/W that allowed the fungi to use the fibres as the secondary carbon source. After 72 h, crude fibre increased by 54.2% in BC/GJ and by 24.8% in BC/W. Starch content was significantly lower (p\u0026thinsp;=\u0026thinsp;0.00) for BC/W at 24 h (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eII, a) due to a faster metabolic activity during the first hours of the fermentation, as seen by the higher free sugar content at the same time point as a result of the enzymatic hydrolysis of starch into smaller oligosaccharides and monosaccharides. After 48 and 72 h, the free sugar content is reduced as monosaccharides are used as the main carbon source for fungal growth and transformed into water and CO\u003csub\u003e2\u003c/sub\u003e. After 72 h, the starch content is reduced by 76.8% in BC/GJ and by 76.5% in BC/W. Ash content was significantly higher (p\u0026thinsp;=\u0026thinsp;0.00) for BC/GJ in all time points likely due to the added minerals, such as phosphorus, potassium, calcium and magnesium, present in GJ, as reported previously in the literature\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. At the end of the SSF, the ratio of CP to starch increased from 0.3 to 2.3 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) which, similar to the changes in crude protein, was significantly different (p\u0026thinsp;=\u0026thinsp;0.00) across time but not significantly different (p\u0026thinsp;=\u0026thinsp;0.39) between the experimental treatments (\u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). As a whole, these results indicated the feasibility of adding forage crop extracts to the SSF process and led to the increasing levels of forage crop protein of the next set of experiments. These changes induced modifications to the flavour profile of the substrates, particularly in BC/GJ, as the material developed sweet notes reminiscent of pineapple and lost all notes related to the green smell of grass, as it was corroborated in informal sensory tests. After 72 h, the senescence of \u003cem\u003eRmo\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI) developed undesirable pungent smells akin to the growth of the black bread mould \u003cem\u003eR. stolonifer\u003c/em\u003e, the most common household bread mould. SPME GC-MS olfactory analysis was carried out over SSF samples to corroborate these findings, but a complete analysis was outside the scope of this publication. Another manuscript detailing these findings is under preparation.\u003c/p\u003e\n\u003cp\u003eThe composition of the BC/GJ/GS samples (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb) was influenced by the amount of GS added, as higher GS content resulted in a lower concentration of starch and a higher concentration of CP in the substrate, while maintaining the same initial moisture. This led to statistical differences for all components (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between experimental treatments and across time, except for crude fibre between the experimental treatments (p\u0026thinsp;=\u0026thinsp;0.20), and significant interactions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the factors for crude protein, free sugars and starch (\u003cstrong\u003eTable S2\u003c/strong\u003e). As the starch content decreased to 10.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8% DM after 72 h from initial contents of 51.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1% DM to 15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7% DM at 0 h, the rest of the components were concentrated. After 72 h, only crude protein and free sugar content were statistically different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) among treatments, while crude fibre, starch and ash content were similar (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). At this point, crude protein content increased by 67.3% in BC/GJ/GS CP 19% DM. Higher GS levels resulted in lower CP content increases after SSF, achieving a 17.8% increase at BC/GJ/GS CP 29% DM. The ratio of CP to starch content was significantly higher (p\u0026thinsp;=\u0026thinsp;0.00) in BC/GJ/GS CP 27% and 29% DM after 72 h (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb) than in the other treatments with GS. As with the BC/W and BC/GJ experiments, the change in the free sugar content also induced changes to the substrate aroma, reducing the characteristic grassy smell, as noticed after informal testing.\u003c/p\u003e\n\u003cp\u003eAt 72h the CP composition of all the fermented substrates were statistically higher (p\u0026thinsp;=\u0026thinsp;0.00) than those of unfermented BC (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, time 0, BC\u0026thinsp;+\u0026thinsp;W) and had a maximum increase in CP of 113.1% in BC/GJ/GS CP 27% DM mirrored by a significant decrease in starch content (p\u0026thinsp;=\u0026thinsp;0.00) of 89.6% (\u003cstrong\u003eTable S3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAmino acid profile analysis\u003c/p\u003e\n\u003cp\u003eEssential (EAA) and non-essential (NEAA) amino acids were analysed by HPLC to evaluate the transformation of the amino acid profile of the BC substrates over time. All EAA, except for tryptophan, were quantified and their concentrations are shown as a coefficient of the crude protein content (g of amino acid / 100 g of CP), as well as the sum of the total amino acids (AA), sum of EAA, sum of NEAA, the percentage of EAA over total AA and NEAA over total AA (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The biotransformation of the protein of the substrate, as an effect of the metabolism of \u003cem\u003eRmo\u003c/em\u003e during the SSF, led to improved amino acid profiles.\u003c/p\u003e\n\u003cp\u003eIn all experimental units, although to a lesser extent in the GS experiments due to the lower percent composition of BC in the substrates, glutamic acid was the AA with the highest concentration at 0 h (from 11.81 to 27.03 g/100 g CP), as this is the predominant AA in wheat protein. Whilst this AA contributes to a pleasing savoury flavour, it is not nutritionally desirable as it is a NEAA. Coupled with the low concentration of lysine versus the FAO nutritional guidelines\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, wheat protein as a whole has a low protein digestibility-corrected amino acid score (PDCAAS) of 0.42\u003csup\u003e37\u003c/sup\u003e and thus not considered a high quality protein for human nutrition. The positive effect of fungal metabolism over the AA profile can be seen in the BC/W SSF (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) with the AA concentrations that were significantly affected by time (\u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). The percentage of EAA over the total AA increased from 30.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5% to 36.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5%, a significant 18.0% increase (p\u0026thinsp;=\u0026thinsp;0.00) after 72 h. At this latter time point, the AA with the greatest significant increases (p\u0026thinsp;=\u0026thinsp;0.00) over time were threonine (19.3%), alanine (112.9%) and aspartic acid (91.4%) while the AA with the greatest significant decreases (p\u0026thinsp;=\u0026thinsp;0.00) were glutamic acid (-47.4%) and proline (-38.1%) (\u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). The bioconversion of certain NEAA, such as glutamic acid, has been studied via metabolic pathways such as the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\alpha\\)\u003c/span\u003e\u003c/span\u003e-aminoadipate pathway, where glutamic acid is the primary amine source for the production of lysine, which selectively allows fungal metabolism to increase the composition of EAA by depleting NEAA\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The variation in total AA was not significant (p\u0026thinsp;=\u0026thinsp;0.38) over time, but its tendency to decrease is potentially due to the continuous cleaving of substrate protein from fungal proteases as time progresses (\u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). Decreasing AA as a percentage of crude protein implies a reduction in the actual functional protein, thus just increasing the value of CP is not enough to consider the valorisation of the substrate. Comparing BC/GJ with BC/W (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) yields few differences in the AA profile. The relatively slower fermentation of BC/GJ (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) produces a maximum percentage of EAA at 48 h (36.4%), instead of 72 h (34.0%) as in BC/W, but the total sum of AA is otherwise similar (p\u0026thinsp;=\u0026thinsp;0.07). Comparing the contents of AA after 72 h of BC/GJ with those of BC/W, BC/GJ had a higher content of phenylalanine, threonine, valine, alanine, glycine, proline, serine, and tyrosine (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eSignificant differences that show the effect of forage protein over the AA profile can be seen in the experiments with BC/GJ/GS (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Increasing additions of GS not only reduces the glutamic acid content at 0 h, but also reduces proline and increases all EAA. Of particular relevance to the nutritional quality of BC, the increase of isoleucine, leucine, lysine and methionine, all present in \u003cem\u003eL. perenne\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, greatly covers the AA deficiencies of wheat. Also, at 0 h, the sum of total AA increased from 67.03\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2 at the lowest level of GS up to 99.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 g/100 g CP at the highest, a 47.8% increase, and the percentage of EAA increased from 28.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 up to 42.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0%, a 48.6% increase. After 72 h of SSF, the sum of total AA significantly decreased (p\u0026thinsp;=\u0026thinsp;0.00) in all experiments between 4.7 and 27.8%, primarily due to the decrease of NEAA, while the percentage of EAA increased by 48.6% at the lowest level of GS and decreased by 8.3% at the highest level of GS. While all EAA were affected throughout the SSF by the addition of GS (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), only histidine, lysine, phenylalanine and threonine were affected by the interaction of both the addition of GS and time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), showing the specific metabolic activity of \u003cem\u003eRmo\u003c/em\u003e towards certain AA of interest, such as lysine (\u003cstrong\u003eTable S2\u003c/strong\u003e). At 72 h the AA profile of the substrates with added GS were significantly different (p\u0026thinsp;=\u0026thinsp;0.00) than those of unfermented BC (\u003cstrong\u003eTable S3\u003c/strong\u003e). The SSF with BC/GJ/GS CP 29% DM had the highest sum of total AA, a 23.4% increase in comparison to unfermented BC, while the SSF with BC/GJ/GS CP 27% DM had the highest percentage composition of EAA, a 54.5% increase in comparison to unfermented BC.\u003c/p\u003e\n\u003cp\u003eThe change of each AA after the SSF varied considerably between each level of GS, likely in response to complex shifts in the metabolic pathways taken by \u003cem\u003eRmo\u003c/em\u003e during SSF, but local maxima and minima can be identified. For instance, the greatest increase in histidine occurs in BC/GJ/GS CP 23% DM (115.4%), of lysine in CP 29% DM (77.8%) and of threonine in CP 19% DM (32.9%), which opens the possibility of targeting the concentration of particular AA at different substrate compositions. Alongside the changes in the proximal composition (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb), this shows that supplementing the CP content of BC with GS results in similar levels of CP (32.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5%) after 72 h of SSF while increasing both the composition of total AA and the percentage composition of EAA.\u003c/p\u003e\n\u003cp\u003eThe impact of these changes for food applications can be further seen in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, which shows the EAA reference ratios calculated against the FAO nutritional guidelines for adults\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Currently, calculation of the nutritional quality of protein is performed via the PDCAAS method, which multiplies the lowest reference ratio from the EAA list with the in-vitro digestibility of the protein. This method undervalues proteins that are deficient in particular AA, such as lysine in wheat, and ignores AA far in excess of the reference. The reference ratios of the EAA in the SSF substrates after 72 h of fermentation increase compared to those of unfermented BC, with the exception of isoleucine, leucine and lysine in BC/GJ/GS 19\u0026ndash;27% DM CP and of methionine\u0026thinsp;+\u0026thinsp;cysteine in BC/W. As neither tryptophan nor the changes in the digestibility of the protein were quantified a PDCAAS could not be calculated, but the ratios suggest that BC/GJ/GS 29% DM CP would have the highest value, as the lowest reference ratio is still for lysine, but is of 0.88 compared to the 0.65 of unfermented BC. In this same SSF sample, all other reference ratios are over 1.0, with particularly high increments in ratio for histidine (2.78) and phenylalanine\u0026thinsp;+\u0026thinsp;tyrosine (3.97). Reports of the changes in the protein digestibility after SSF with \u003cem\u003eRhizopus\u003c/em\u003e sp. are mixed in the literature\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and could be affected by the phenolic content of PRG and the changes in the free amino acid content of the substrate after SSF.\u003c/p\u003e\n\u003cp\u003eThis research establishes the viability and nutritional potential of combining forage crops and bakery surplus to reduce waste, improve circularity within the food industry and upgrade non-conventional crops for further development as human food. As such, this provides novel insights for the potential of other combinations of substrates to be fermented with \u003cem\u003eRmo\u003c/em\u003e, or other food-safe filamentous fungi used in SSF, such as \u003cem\u003eAspergillus\u003c/em\u003e sp. and \u003cem\u003eNeurospora\u003c/em\u003e sp. and explore the changes in the nutritional quality and sensory profile. Industrially, this provides the agricultural sector an additional route to valorise their crops through a biorefinery, and the food manufacturing sector information to develop novel alternatives for animal proteins with lower environmental impacts, which would reduce food waste, increase food security, and decarbonize the food system. The resulting fermented products could thus be explored as novel foods, before being dried and milled, or the processed protein-rich powder incorporated in the preparation of wheat-based staple foods such as bread, cookies, pasta, pastries and cakes, by replacing fractions of wheat flour, which requires further research.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eRaw materials\u003c/p\u003e \u003cp\u003eAcetonitrile, formic acid, ammonium formate, ethanol and sodium hydroxide were purchased from Fischer Scientific UK Ltd (Leicestershire, UK). Phosphoric acid, phenol, DL-norvaline, potato dextrose agar and tween 80 were purchased from Merck Life Science UK Ltd (Dorset, UK). Anhydrous acetonitrile, hydrochloric acid, borate buffer and amino acid standard H were purchased from ThermoFischer Scientific Ltd (Paisley, UK). 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was purchased from Synchem UG \u0026amp; Co. KG (Altenburg, Germany). Total starch analysis kit was purchased from Megazyme Ltd (Wicklow, Ireland). Unless specified, all this study's chemicals, solvents and reagents were of at least analytical grade. For HPLC, the AccQ-Tag Amino acids C18 column was purchased from Waters Ltd (Wilmslow, UK), and the REZEX-ROA Organic acid H+ (8%) Ion exclusion column was purchased from Phenomenex Inc (CA, US). Bread crusts (BC) from surplus wheat flour loaf bread were provided by Bradgate Bakery (a division of Samworth Brothers Ltd, Leicestershire, UK) and manually cut to approximately 1x1x1 cm cubes.\u003c/p\u003e \u003cp\u003ePerennial ryegrass harvesting and pilot-scale processing\u003c/p\u003e \u003cp\u003ePerennial ryegrass (\u003cem\u003eLolium perenne\u003c/em\u003e) was seeded in 2021 in a farmed experimental plot at Aberystwyth University in Aberystwyth, UK and harvested in June 2022. 1 ton of freshly harvested PRG was screw pressed at pilot scale in a CP-10 screw press (Vincent corporation, FL, US) to obtain 410 L of GJ with a pH of 5.62 (adjusted to pH 3.5 with H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) and frozen at -20\u0026deg;C in 2 L aliquots to avoid the growth of undesired microorganisms. The resulting GJ had a DM content of 4.98% and CP content of 16.0% DM. 32 L of GJ were clarified using a GLE continuous centrifuge (Carl Padberg Zemtrofugenbau GmbH, Lahr, Germany) at 38000 x g to obtain 800 g of wet protein-rich precipitate with a DM content of 27.3%, which was freeze-dried to a GS with final DM content of 85.0% and CP content of 34.9% DM.\u003c/p\u003e \u003cp\u003eMicroorganism and inoculum preparation\u003c/p\u003e \u003cp\u003e \u003cem\u003eRmo\u003c/em\u003e spores were purchased from fermentationculture.eu and preserved in 10% glycerol solution at -80\u0026deg;C and in potato dextrose agar (PDA) plates at 32\u0026deg;C. After 5 days of growth at 32\u0026deg;C \u003cem\u003eRmo\u003c/em\u003e spores were harvested from PDA plates in 50 mL sterile distilled water (0.03% tween 80) and counted in a Neubauer chamber to approximately \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(1\\times {10}^{7}\\)\u003c/span\u003e\u003c/span\u003e spores/mL and SSFs were inoculated at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(2\\times {10}^{5}\\)\u003c/span\u003e\u003c/span\u003e spores/g substrate.\u003c/p\u003e \u003cp\u003eSolid-state fermentation\u003c/p\u003e \u003cp\u003eSSF experiments were conducted in sterile 100 mm diameter Petri dishes. The experiments were divided into two sequential sections: firstly, 25 g of BC were adjusted to a moisture content of 56% (v/w DM) with either sterile distilled water (W) or GJ and fermented for 24, 48 or 72 h. These experiments were done in triplicate for a total of n\u0026thinsp;=\u0026thinsp;24 samples. Secondly, 25 g of BC were adjusted to a moisture content of 56% (v/w DM) with GJ, supplemented with GS to final substrate CP contents of 19%, 20%, 23%, 27% or 29% DM, and fermented for 72 h. These experiments were done in duplicate for a total of n\u0026thinsp;=\u0026thinsp;24 samples. For each fermentation, 40 g of mixed substrate (BC with W/GJ/GS), at pH 3.5 (adjusted with H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), covered with lids but not closed hermetically to enable airflow, were incubated at 32\u0026deg;C, having been weighed and photographed before and after the SSF. All fermented samples were dried in a vacuum oven at 45\u0026deg;C, milled to a fine powder and then stored at 4\u0026deg;C for further analysis.\u003c/p\u003e \u003cp\u003eChemical composition analysis\u003c/p\u003e \u003cp\u003eThe moisture content of the samples was determined according to AOAC method 930.15\u003csup\u003e41\u003c/sup\u003e. Ash content was determined according to AOAC method 942.05\u003csup\u003e41\u003c/sup\u003e. Crude protein content was determined using the Dumas combustion method in a Vario MAX Cube CN analyser (Elementar, Stockport, UK). A conversion factor of 6.25 was used to convert total nitrogen to crude protein content. Crude fibre content was determined according to the AOCS method Ba 6a-05\u003csup\u003e42\u003c/sup\u003e in an ANKOM A2000 fibre analyser (ANKOM Technology, NY, US). Free sugars (glucose, fructose and sucrose) were determined by mixing 200 mg of sample with 1 mL of 10% ethanol solution. Sugars were extracted by shaking at room temperature (25\u0026deg;C) for 1 h, followed by centrifugation at 21000 x g for 10 min with the supernatants stored at 4\u0026deg;C and the pellets discarded. The supernatants containing free sugars were filtered through a 0.22 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003em membrane and quantified by HPLC in a REZEX-ROA Organic acid H+ (8%) Ion exclusion column with a refractive index (RI) detector. Total starch content was determined according to the AOAC method 996.11\u003csup\u003e41\u003c/sup\u003e using an amyloglucosidase/\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\alpha }\\)\u003c/span\u003e\u003c/span\u003e-amylase assay kit (Megazyme, Wicklow, Ireland). The supernatants containing hydrolysed starch were filtered through a 0.22 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003em membrane and quantified by HPLC in a REZEX-ROA Organic acid H+ (8%) Ion exclusion column with a refractive index (RI) detector. Measurements were done once per experimental replicate. All values are reported as percent dry matter basis (% DM).\u003c/p\u003e \u003cp\u003eAmino acid profile analysis\u003c/p\u003e \u003cp\u003eTotal AAs were prepared by mixing 200 mg of milled, dry SSF samples with 5 mL of 6 M HCl (0.1% phenol) in 15 mL glass tubes, degassed and sealed under an N\u003csub\u003e2\u003c/sub\u003e stream, and incubated for 24 h at 110\u0026deg;C and allowed to cool to room temperature. The hydrolysates were adjusted with 6 M NaOH to pH 6\u0026ndash;8, made up to a final volume of 50 mL with water, and then centrifuged at 21000 x g for 10 min, with the supernatant filtered through a 0.22 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003em membrane and added in a 1:1 ratio to a 250 pmol/\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL solution of DL-norvaline as an internal standard.\u003c/p\u003e \u003cp\u003ePre-column derivatisation of amino acids with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was performed in accordance with Reverter, et al. \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Briefly, 70 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL of borate buffer was added to 10 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL of AA hydrolysate, or amino acid standards, and 20 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL of AQC solution (4 mg AQC in 1 mL anhydrous acetonitrile). After 1 min incubation at room temperature, the contents were transferred to an autosampler vial and capped with a silicone-lined septum. Derivatised samples were quantified by HPLC using an AccQ-Tag Amino acids C18 column with variable wavelength (VWD) (260 nm detection) and fluorescence (FLD) (266 nm emission and 473 nm detection) detectors. Mobile phases were composed of water (solvent A), acetonitrile (solvent B) and 50 mM ammonium formate (adjusted to pH 2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 with formic acid) (solvent C). Elution was carried out as follows: 0-0.5 min 0\u0026ndash;1% solvent B and 100\u0026thinsp;\u0026minus;\u0026thinsp;99% solvent C, 0.5\u0026ndash;15 min 1\u0026ndash;4% solvent B and 99\u0026thinsp;\u0026minus;\u0026thinsp;96% solvent C, 15\u0026ndash;23 min 4\u0026ndash;5% solvent B and 96\u0026thinsp;\u0026minus;\u0026thinsp;95% solvent C, 23\u0026ndash;24 min 5\u0026ndash;9% solvent B and 95\u0026thinsp;\u0026minus;\u0026thinsp;91% solvent C, 24\u0026ndash;28 min 9\u0026ndash;14% solvent B and 91\u0026thinsp;\u0026minus;\u0026thinsp;86% solvent C, 28\u0026ndash;45 min 14\u0026ndash;17% solvent B and 86\u0026thinsp;\u0026minus;\u0026thinsp;83% solvent C, 45\u0026ndash;55 min 17\u0026ndash;60% solvent B and 83\u0026thinsp;\u0026minus;\u0026thinsp;0% solvent C. The flow rate was 1 mL/min, and the injection volume was 10 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL. The AA in the samples were analysed according to retention time and peak area.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe analytical data was analysed by two-tailed analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s HSD post-hoc test using IBM SPSS v 28 statistical software (IBM UK Ltd, Hampshire, UK). The effect of the experimental treatments was analysed with a one-way ANOVA by comparing the results from each measurement at each time point (0, 24, 48 and 72 h) in two groups, BC/W and BC/GJ, and the different levels of BC/GJ/GS. The effect of time in the fermentation and the interaction between the experimental treatment and fermentation time, was analysed by a two-way ANOVA in two separate groups, BC/W and BC/GJ (supplementary \u003cb\u003eTable S1\u003c/b\u003e), and the different levels of BC/GJ/GS (supplemental \u003cb\u003eTable S2\u003c/b\u003e). Lastly, all experimental treatments at 72 h of SSF and those of unfermented BC were compared to identify the significant, most impactful changes with a one-way ANOVA followed by Tukey\u0026rsquo;s HSD post-hoc test (supplementary \u003cb\u003eTable S3\u003c/b\u003e). The results were expressed as mean value \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\pm\\)\u003c/span\u003e\u003c/span\u003e standard error. A statistical significance level (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\alpha }\\)\u003c/span\u003e\u003c/span\u003e 0.05) was used to analyse the results.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings reported herein are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the BBSRC FoodBiosystems DTP committee and the staff of IBERS, Aberystwyth University for their support. This research was funded by the BBSRC, UKRI doctoral training grant no: BB/T008776/1. The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Aberystwyth, SY23 3EE, UK\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJuan Felipe Sandoval, Joe Gallagher \u0026amp; David N. Bryant\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Faculty of Pharmacy, Universitat de Val\u0026egrave;ncia, Avda.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eVicent Andr\u0026eacute;s Estell\u0026eacute;s, s/n, 46100 Burjassot, Val\u0026egrave;ncia, Spain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJulia Rodriguez-Garcia\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSamworth Brothers Limited, Leicestershire, LE13 1GA, UK\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKerry Whiteside\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e*\u003c/sup\u003eCorresponding author. Tel: +44 07432487541. E-mail: [email protected]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors designed this study, reviewed and approved the manuscript. JG, JRG, KW and DNB acquired the funding and supervised the research. \u0026nbsp;JFS, JG, JRG and DNB contributed to data analysis. JFS conducted the experiments, acquired and curated the data and wrote the original manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no financial or non-financial competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eReynolds, M. P. \u0026amp; Braun, H.-J. \u003cem\u003eWheat Improvement: Food Security in a Changing Climate\u003c/em\u003e. (Springer Nature, 2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFAOSTAT statistical database ([Rome]: FAO, c1997-, 2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNarisetty, V. \u003cem\u003eet al.\u003c/em\u003e Recycling bread waste into chemical building blocks using a circular biorefining approach. 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Determination of free amino acids in pig plasma by precolumn derivatization with 6-N-aminoquinolyl-N-hydroxysuccinimidyl carbamate and high-performance liquid chromatography. Journal of Chromatography B: Biomedical Sciences and Applications 696, 1\u0026ndash;8 (1997). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/S0378-4347(97)00217-X\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/S0378-4347(97)00217-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Total amino acid profile [g/100g CP] from bread crusts (BC)/water (W) and BC/green juice (GJ) at 0, 24, 48 and 72 h of solid-state fermentation (SSF).\u0026nbsp;Results are expressed as the mean value\u0026nbsp;\u0026plusmn; a standard error.\u0026nbsp;Data with different letters in the same time point are significantly different (p \u0026lt; 0.05) between BC/W and BC/GJ, following Tukey\u0026acute;s HSD post-hoc test. Data with the same letters are statistically similar.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubstrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eBC/W\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eBC/GJ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eComposition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e24 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e48 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e72 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e24 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e48 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e72 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHistidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.08 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.76 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.87 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.90 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.61 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.61 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.85 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.98 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIsoleucine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.47 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.72 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.00 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.78 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.28 \u0026plusmn; 0.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.25 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.85 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.68 \u0026plusmn; 0.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLeucine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.00 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.62 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.74 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.42 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.58 \u0026plusmn; 0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.04 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.17 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.50 \u0026plusmn; 0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLysine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.94 \u0026plusmn; 1.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.74 \u0026plusmn; 1.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.62 \u0026plusmn; 1.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.54 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.18 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.77 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.39 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.17 \u0026plusmn; 0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMethionine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.30 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.39 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.25 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.81 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.02 \u0026plusmn; 0.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.65 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.70 \u0026plusmn; 0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.19 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePhenylalanine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.18 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.93 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.80 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.44 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.55 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.63 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.94 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.38 \u0026plusmn; 0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eThreonine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.18 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.42 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.69 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.60 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.57 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.78 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.05 \u0026plusmn; 0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eValine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.28 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.87 \u0026plusmn; 0.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.35 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.97 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.03 \u0026plusmn; 0.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.70 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.33 \u0026plusmn; 0.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.59 \u0026plusmn; 0.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNEAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAlanine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.71 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.82 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.94 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.77 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.28 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.86 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.12 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.62 \u0026plusmn; 0.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAspartic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.18 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.02 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.81 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.08 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.55 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.17 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.91 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.17 \u0026plusmn; 0.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCysteine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.28 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.18 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.26 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.30 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.30 \u0026plusmn; 0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.18 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.37 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGlutamic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25.58 \u0026plusmn; 0.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18.68 \u0026plusmn; 1.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17.20 \u0026plusmn; 0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13.45 \u0026plusmn; 0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e27.03 \u0026plusmn; 3.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19.99 \u0026plusmn; 2.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.28 \u0026plusmn; 1.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15.35 \u0026plusmn; 2.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGlycine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.97 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.04 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.37 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.18 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.85 \u0026plusmn; 0.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.76 \u0026plusmn; 0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.83 \u0026plusmn; 0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.76 \u0026plusmn; 0.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eProline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.72 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.02 \u0026plusmn; 0.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.76 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.40 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.87 \u0026plusmn; 0.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.94 \u0026plusmn; 0.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.27 \u0026plusmn; 0.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.17 \u0026plusmn; 0.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSerine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.19 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.79 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.82 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.65 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.26 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.18 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.89 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.56 \u0026plusmn; 0.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTyrosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.73 \u0026plusmn; 0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.30 \u0026plusmn; 1.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.00 \u0026plusmn; 0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.62 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.64 \u0026plusmn; 0.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.34 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.64 \u0026plusmn; 0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.61 \u0026plusmn; 0.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eArginine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.70 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.38 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.84 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.76 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.56 \u0026plusmn; 0.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.86 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.42 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.78 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSum AA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76.49 \u0026plusmn; 2.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e72.66 \u0026plusmn; 8.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75.32 \u0026plusmn; 5.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e67.65 \u0026plusmn; 0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e70.59 \u0026plusmn; 7.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e69.51 \u0026plusmn; 5.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e60.55 \u0026plusmn; 4.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e69.93 \u0026plusmn; 7.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSum EAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e23.43 \u0026plusmn; 1.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25.45 \u0026plusmn; 4.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e26.33 \u0026plusmn; 2.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24.45 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19.26 \u0026plusmn; 2.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e22.22 \u0026plusmn; 1.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e22.03 \u0026plusmn; 1.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e23.54 \u0026plusmn; 1.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSum NEAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e53.06 \u0026plusmn; 1.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e47.21 \u0026plusmn; 4.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e48.99 \u0026plusmn; 2.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.20 \u0026plusmn; 0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e51.34 \u0026plusmn; 5.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e47.29 \u0026plusmn; 4.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e38.52 \u0026plusmn; 3.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46.39 \u0026plusmn; 5.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e% EAA/AA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30.63 \u0026plusmn; 1.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34.63 \u0026plusmn; 0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34.80 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e36.15 \u0026plusmn; 1.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e27.07 \u0026plusmn; 1.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e32.03 \u0026plusmn; 1.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e36.38 \u0026plusmn; 1.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34.00 \u0026plusmn; 0.5\u003csup\u003ea\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e% NEAA/AA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e69.37 \u0026plusmn; 1.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e65.37 \u0026plusmn; 0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e65.20 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e63.85 \u0026plusmn; 1.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e72.93 \u0026plusmn; 1.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e67.97 \u0026plusmn; 1.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e63.62 \u0026plusmn; 1.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e66.00 \u0026plusmn; 0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003e\n \u003cp\u003eCP: Crude protein, AA: Amino acids EAA: Essential amino acids, NEAA: Non-essential amino acids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Total amino acid profile [g/100g CP] from bread crusts (BC)/green juice (GJ)/dry green solids (GS) to CP 19%, 20%, 23%, 27% and 29% DM at 0 and 72 h of solid-state fermentation (SSF). Results are expressed as the mean value \u0026plusmn; a standard error. Data with different letters in the same time point are significantly different (p \u0026lt; 0.05) between the different levels of GS, following Tukey\u0026acute;s HSD post-hoc test. Data with the same letters are statistically similar.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.34020618556701%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eCP 0 h [%DM]\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e19%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e20%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e23%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e27%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e29%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003e\u003cstrong\u003eComposition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e\u003cstrong\u003e72 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e\u003cstrong\u003e72 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e\u003cstrong\u003e72 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e\u003cstrong\u003e72 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e\u003cstrong\u003e72 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003e\u003cstrong\u003eEAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003eHistidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e1.18 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e1.36 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e1.87 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e2.47 \u0026plusmn; 0.0\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e2.40 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e5.18 \u0026plusmn; 0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e3.37 \u0026plusmn; 1.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e2.72 \u0026plusmn; 0.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e6.55 \u0026plusmn; 0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e4.17 \u0026plusmn; 0.4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003eIsoleucine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e1.57 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e1.86 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e2.53 \u0026plusmn; 0.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e2.19 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e2.94 \u0026plusmn; 0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e2.45 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e2.81 \u0026plusmn; 0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e2.60 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e3.70 \u0026plusmn; 0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e3.62 \u0026plusmn; 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0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e3.86 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e5.07 \u0026plusmn; 0.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e5.84 \u0026plusmn; 0.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003eAspartic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e4.52 \u0026plusmn; 1.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e6.76 \u0026plusmn; 0.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n 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3.0\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e11.49 \u0026plusmn; 1.1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e25.08 \u0026plusmn; 1.8\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e11.10 \u0026plusmn; 0.2\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e20.44 \u0026plusmn; 0.5\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e8.92 \u0026plusmn; 0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e13.05 \u0026plusmn; 2.0\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003eProline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e6.27 \u0026plusmn; 0.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e3.77 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e7.52 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e3.90 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e6.97 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n 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0.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e5.45 \u0026plusmn; 0.0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e4.51 \u0026plusmn; 0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003eTyrosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e3.37 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e2.94 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e3.99 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e2.91 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e4.53 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e4.52 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e5.17 \u0026plusmn; 0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e5.24 \u0026plusmn; 1.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e5.18 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e5.24 \u0026plusmn; 0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003eArginine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e2.29 \u0026plusmn; 0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e3.30 \u0026plusmn; 0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e4.20 \u0026plusmn; 0.5\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e4.11 \u0026plusmn; 0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e5.24 \u0026plusmn; 0.4\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e8.34 \u0026plusmn; 0.3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e6.15 \u0026plusmn; 1.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e6.70 \u0026plusmn; 0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e10.97 \u0026plusmn; 0.0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e7.60 \u0026plusmn; 0.3\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSum AA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e67.03 \u0026plusmn; 7.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e57.02 \u0026plusmn; 0.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e85.89 \u0026plusmn; 0.4\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e62.01 \u0026plusmn; 1.7\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e87.51 \u0026plusmn; 1.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e80.22 \u0026plusmn; 2.6\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e79.50 \u0026plusmn; 2.4\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e69.28 \u0026plusmn; 2.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e99.05 \u0026plusmn; 0.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e94.42 \u0026plusmn; 6.4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSum EAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e19.15 \u0026plusmn; 1.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e18.95 \u0026plusmn; 0.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e26.57 \u0026plusmn; 0.8\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e22.05 \u0026plusmn; 0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e30.16 \u0026plusmn; 1.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e31.00 \u0026plusmn; 0.7\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e30.90 \u0026plusmn; 2.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e29.00 \u0026plusmn; 1.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e42.29 \u0026plusmn; 0.3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e36.92 \u0026plusmn; 1.8\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSum NEAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e47.88 \u0026plusmn; 6.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e38.07 \u0026plusmn; 1.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e59.31 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e39.96 \u0026plusmn; 1.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e57.35 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e49.22 \u0026plusmn; 3.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e48.60 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e40.28 \u0026plusmn; 0.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e56.77 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e57.50 \u0026plusmn; 4.6\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003e\u003cstrong\u003e% EAA/AA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e28.72 \u0026plusmn; 1.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e33.24 \u0026plusmn; 1.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e30.94 \u0026plusmn; 1.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e35.56 \u0026plusmn; 0.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e34.45 \u0026plusmn; 0.5\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e38.71 \u0026plusmn; 2.1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e38.80 \u0026plusmn; 2.4\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e41.83 \u0026plusmn; 1.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e42.69 \u0026plusmn; 0.0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e39.15 \u0026plusmn; 0.7\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.956521739130435%\"\u003e\n \u003cp\u003e\u003cstrong\u003e% NEAA/AA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e71.28 \u0026plusmn; 1.4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e66.76 \u0026plusmn; 1.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e69.06 \u0026plusmn; 1.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e64.44 \u0026plusmn; 0.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.782608695652174%\"\u003e\n \u003cp\u003e65.55 \u0026plusmn; 0.5\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e61.29 \u0026plusmn; 2.1\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e61.20 \u0026plusmn; 2.4\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e58.17 \u0026plusmn; 1.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e57.31 \u0026plusmn; 0.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\"\u003e\n \u003cp\u003e60.85 \u0026plusmn; 0.7\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"11\"\u003e\n \u003cp\u003eCP: Crude protein, DM: Dry matter, AA: Amino acids EAA: Essential amino acids, NEAA: Non-essential amino acids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Essential amino acid (EAA) reference ratios calculated against the FAO nutritional guidelines (g/100g CP) for adults for unfermented bread crusts (BC), and solid-state fermentation (SSF) substrates BC/water (W), BC/green juice (GJ), BC/GJ/green solids (GS) to CP 19%, 20%, 23%, 27% and 29% DM at 72 h.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eEAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eFAO requirement\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003eBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubstrate after 72 h SSF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBC/W\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBC/GJ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003eBC/GJ/GS to % DM CP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e19%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e23%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e27%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e29%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHistidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIsoleucine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLeucine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLysine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eThreonine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eValine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMethionine +\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCysteine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePhenylalanine +\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTyrosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTryptophan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e\n \u003cp\u003eCP: Crude protein, DM: Dry matter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3973183/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3973183/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSolid-state fermentation (SSF) is a sustainable method to convert food waste and plant biomass into novel foods for human consumption. Surplus bread crusts (BC) have the structural capacity to serve as SSF scaffold, and their nutritional value could be increased in combination with perennial ryegrass (PRG), a biorefining feedstock with high-quality protein but an unpleasant sensory profile. SSF with \u003cem\u003eRhizopus oligosporus\u003c/em\u003e was investigated with these substrates to determine if the overall nutritional value could be increased. The BC-PRG SSFs were conducted for up to 72 h, over which time the starch content had decreased by up to 89.6%, the crude protein content increased by up to 113.1%, and the essential amino acid content increased by up to 54.5%. The BC-PRG SSF demonstrated that this process could potentially valorise BC and PRG, both widely available but underexplored substrates, offering feedstock for alternative protein sources.\u003c/p\u003e","manuscriptTitle":"Improved Nutritional Value of Surplus Bread and Perennial Ryegrass Extracts Via Solid-State Fermentation (SSF) with Rhizopus oligosporus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-14 08:05:14","doi":"10.21203/rs.3.rs-3973183/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-07-24T10:42:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-13T10:19:39+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-01T09:32:04+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-06-18T09:55:02+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-05-21T07:33:29+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-05-03T07:15:24+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-03-12T05:12:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-21T09:51:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Science of Food","date":"2024-02-20T15:23:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-20T15:23:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d48abbf-6355-4d3f-9ce7-06c76c8f10e4","owner":[],"postedDate":"March 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29339040,"name":"Biological sciences/Microbiology/Applied microbiology"},{"id":29339041,"name":"Biological sciences/Biochemistry/Proteins"}],"tags":[],"updatedAt":"2024-11-17T08:05:39+00:00","versionOfRecord":{"articleIdentity":"rs-3973183","link":"https://doi.org/10.1038/s41538-024-00338-y","journal":{"identity":"npj-science-of-food","isVorOnly":false,"title":"npj Science of Food"},"publishedOn":"2024-11-16 05:00:00","publishedOnDateReadable":"November 16th, 2024"},"versionCreatedAt":"2024-03-14 08:05:14","video":"","vorDoi":"10.1038/s41538-024-00338-y","vorDoiUrl":"https://doi.org/10.1038/s41538-024-00338-y","workflowStages":[]},"version":"v1","identity":"rs-3973183","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3973183","identity":"rs-3973183","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}