Fish waste valorization: protein hydrolysate as sustainable nitrogen and nutrients for production of carotenogenic yeasts biomass.

doi:10.21203/rs.3.rs-5146845/v1

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Fish waste valorization: protein hydrolysate as sustainable nitrogen and nutrients for production of carotenogenic yeasts biomass.

https://doi.org/10.21203/rs.3.rs-5146845/v1

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Global fisheries and aquaculture production reached a record of 178 Mt in 2020. It is forecast that production will rise to 200 Mt by 2029, representing an increase of 25 Mt (14%). However, challenges arise with this increase like fish waste that comprising about two-thirds of total fish production and leading to economic and environmental concerns. The emergence of the bio-based circular economy is explored as a solution to manage fish wastes sustainably and biorefinery approach plays a central role in transforming industrial by-products into value-added products. An alternative to managing fish wastes is the production of fish protein hydrolysates, which contain proteins, oligopeptides and free amino acid, among other nutritional and functional compounds, with many technological applications. The species utilized in this study to produce fish protein hydrolysates was the chondrichthyan Mustelus schmitti, a specie caught by both artisanal and commercial fisheries in Argentina and Uruguay but in vulnerable exploitation due to its life cycle which compromised its fishery sustainability. In this scenario, complete biomass utilization of the already harvested individuals has been proposed encouraging maximal and sustainable use of M. schmitti fishing resources. Fish protein hydrolysates supplemented with dextrose was used as the sole nutrition source for the growth of a carotenogenic red yeast Rhodotorula glutinis capable of producing carotenoids tolurene and torularhodin. Our results indicate that fish protein hydrolysates from M. schmitti is able to support the growth of R. glutinis as the sole nutritional source and enhance carotenoid production compared with laboratory reference culture media.

Applied & Industrial Microbiology

Food Science & Technology

Biotechnology and Bioengineering

fish protein hydrolysates

Mustelus schmitti

nutrients source

yeast growth

carotenoids

Fish wastes was processed totally to formulate sustainable yeast growth media

Fish protein hydrolysate alone support yeast growth.

Medium based on fish protein hydrolysates improve carotenoid production

The World Fisheries and Aquaculture 2022 document from Food and Agriculture Organization [1] indicate that global fisheries and aquaculture production is at a record high, with a production reaching 178 million tons in 2020. An 89% of total production was used for direct human consumption with a per capita value of 20.2 kg in 2020 and several factors like urbanization and dietary trends among others are projected to drive an increase in aquatic food consumption up to 21.4 kg per capita in 2030. It is imperative to enhance worldwide fisheries management to restore ecosystems, ensuring their health and productivity, and safeguard the sustainable provision of aquatic food in the long run [1]. In line with the increased fish production, the amount of fish waste has also risen. Late estimations indicate about two-thirds of the total amount of fish is discarded as waste, creating economic and environmental concerns [2]. Consequently, there is a pressing need for improved fish waste management to address these concerns and fully capitalize the high-commercial value potential of this biomass. Actually, fish waste has been used for purposes like fish meal, fertilizers, and fish oil production [3], [4], [5], [6], or it is simply discarded [7]. In this context, focus on alternative applications of fish byproducts plays a significant role in promoting economic growth and sustainable development [2].

The bioeconomy is an integral component of the broader circular economy framework, serving as a crucial driver for achieving sustainability goals related to both resource management and environmental conservation [8]. Technological advancements centered on utilizing biomass as a substrate for the production of bio-based products are in line with the principles of a biorefinery approach [9]. Waste-focused biorefineries have gained worldwide attention among researchers for their capacity to transform industrial by-products into value-added products [10].

The fish protein hydrolysates (FPH) has gained significant attention, primarily because of its capacity to enhance protein retrieval and the growing attraction to exploring potential industrial uses for the resulting hydrolysates [11], [12]. The FPHs refers to a blend of proteins fragments achieved by subjecting biomass to hydrolysis, resulting in the formation of oligopeptides and free amino acids (FAA). There are three principal methods to obtain FPH including chemical method, fermentation method and enzymatic method. Especially, the enzymatic method using specific proteases has received great attention [13]. In this process a protease is added to biomass and under specific controlled reaction conditions of temperature, pH, agitation and substrate/enzyme ratio, among others; allow generation of a consistent product [11]. Several functions have been attribute to FPH based of its content of bioactive peptides with antimicrobial, antioxidant, antihypertensive and anticancer properties [2]. Additionally, FPH has been used as a peptone and source of nutrients to growth diverse microorganisms like yeasts and bacteria [14], [15], [16], [17], [18]. In this sense, microbial based platforms have been developed to produce high value products from industrial streams. Microbial valorization also reduces production costs and the treatment costs traditionally associated with waste disposal [19]. An additional factor to add on microbial platforms could be the exploitation of native biodiversity. Nature harbors multiple, yet unknow, species and strains that could perform superior industrial fermentation [20]. Bioprospection to find technological relevant new species and/or strains is of great importance.

Mustelus schmitti Springer 1939 (Carcharhiniformes: Triakidae) narrownose shark is a small-sized demersal coastal specie, commonly named Gatuzo. This species exhibits a wide geographical range encompassing southwestern Atlantic region, spanning from the southern reaches of Brazil (22°S) to southern Argentina (at 47°45′S) [21]. Concentrated populations are found along the coast of Uruguay and the northern Argentine coastal system, where it becomes the focus of attention for both commercial bottom trawl fisheries and artisanal gillnet fishing [22], [23], [24], [25], [26]. This species of shark it is important in fishery landings, since it is captured by the multispecific fleet called "coastal variety" and to a lesser extent as accompanying fauna [27]. Data from last four years (from 2020 to 2023) indicate that commercial catch of this specie reach an average of 2862.1 metric tones [28]. Like others chondrites, M. schmitti is characterized by low fecundity, slow growth rate, late sexual maturity and high longevity. These traits lead to a low reproductive potential and low population growth rate which restrict the capacity of this population to sustain a fishery and/or recover of human impact [29]. M. schmitti it considered critically endangered according to the IUCN Red List of Threatened Species™ [30]. Despite these critical factors, exploitation of M. schmitti persists using only muscle for human consumption and without another commercial use of remaining biomass. To ensure conservation and rational exploitation of this shark it was established a Management Plan for Restoration of Gatuzo in the common fishing zone of Argentine-Uruguay driven by Comisión Técnica Mixta de Frente Marítimo [31]. Actions of this plan include the integral use of gatuzo biomass with focus on biorefinery [32].

In this work, we use FPH obtained from wastes of M. schmitti as a nitrogen and nutrient sustainable source for yeast growth. In order to add value to FPH the selected yeast was a native carotenogenic strain. This strain was obtained from a bioprospecting approach aimed to obtain yeast diversity that coexist with the native and endemic flora of the southeast Buenos Aires Province, Argentine. The selected strain was identified as Rhodotorula glutinis, a carotenogenic strain [33], [34] that produces carotenoids tolurene and torularhodin with great potential for use in industrial applications [35], [36].

2.1. M. schmitti waste processing

Specimens of M. schmitti were captured during the year 2021 under researcher campaign conducted by the Instituto Nacional de Investigation y Desarrollo Pesquero (INIDEP) aboard the RV "Eduardo Holmberg" (INIDEP EH-05-21) in November 2021 within the Buenos Aires Province coastal region (34° S-42° S, 62° W-52° W). Nine frozen individuals of M. schimitti with approximately 40–60 cm of length, were completely eviscerated with additional remotion of muscle and remaining biomass (waste) were divided in two pools from 4–5 individuals and processes independently as sample 1 and sample 2. Wastes were cut into approximately 5 cm³ pieces and milled with a commercial meat miller (Freire SA) with a sieve of 4 mm. Milled biomass was used immediately or stored at -20°C until use.

2.2. Enzymatic hydrolysis

M. schmitii waste (90 gr) was mixed with deionized water in a ratio of 1:2 ^w/_v in 300 mL glass reactor. Temperature was controlled to 60°C ± 1°C, agitation at 60 rpm and pH adjusted to 8 with NaOH 2 M. In this condition, alkaline protease Alcalase 2.4L (Sigma Aldrich) was added (final concentration 1% ^v/_v). Hydrolysis was performed following pHstat method [37] using NaOH 2M to maintaining pH 8 for 50 minutes. At the end of hydrolysis, the pH of hydrolysate was lowered to 5.5 with ortophosphoric acid 85% to inactivate enzymatic action. Hydrolysate was let to set down to separate soluble and insoluble phases by decantation. Soluble fraction was filter using a mesh of 100 µm, centrifugated at 3000 rpm for 20 minutes and aqueous fraction (MsFPH) stores at -20°C until use.

2.3. Proximate composition analysis

Moisture content was determined by oven-drying at 105°C to constant mass [38]. The ash content was determined by calcination of the sample at 550°C to obtain ash of constant mass [39]. Crude proteins were analyzed according to the Kjeldahl method [40]. A factor of 6.25 was used for the conversion from total nitrogen to protein. Non-protein nitrogen content was analyzed according to [41].

2.4. Measurement of soluble protein, carbohydrates and free amino acids

Soluble proteins were quantified by Lowry method [42] and carbohydrates according to [43]. FAA were determinate by High Performance Liquid Chromatography (HPLC). Samples of MsFPH or yeast cell free exhausted medium were centrifuged at 5000 rpm for 20 minutes. The supernatant was treated with 2% of TCA ^w/_v and ethanol 70% ^v/_v in a relation 1:4:1 respectively and incubated during 1 h with shaking [44]. Samples were centrifuged at 10,000 rpm for 20 minutes and supernatant was used for HPLC analysis. The supernatant was derivatized with a Shimadzu® SIL 10AF automatic injector. For this, 50 µL of sample was mixed for 1 min with 200 µl of OPA solution (27 mg of o-phthaldialdehyde + 500 µL of ethanol + 5 mL of 0.1M sodium tetraborate pH 9,5 and 50 µL of 2-mercaptoethanol). The derivatized sample was injected in HPLC with a VP-ODS Shim-pack Shimadzu® C18 column (150 mm x 4.6 mm i.d. x 5µm) and a GVP-ODS Shim-pack Shim-pack Shimadzu® C18 cartridge (10 mm x 4.6 mm i.d.) guard column, which were maintained at 25°C in a Shimadzu® CTO-10AS VP oven. The mobile phase was 0.05 M buffer acetate pH 6.8 (A) and 100% HPLC grade methanol (B), which was filtered with a 0.22 µm membrane. The flow rate was 1 mL.min^− 1 using two Shimadzu® LC20AT pumps. Solvent gradient started with 19% B, from 19–28.15% in 5 min; from 28.15–50% in 5 min, stays at 50% for 2 min; from 50–60.5% in 14.5 min; from 60.5–19% in 5 min. Detection was performed by fluorescence at excitation wavelength of 340 nm and emission of 450 nm. The amino acids were identified and quantified by comparison with retention times and areas of pure standards mix (Sigma AA9906).

2.5. Gradient SDS-PAGE

For the evaluation of the hydrolysis process, molecular size of the peptides was determinate by gradient SDS-PAGE (10%-18% acrylamide, 0,5%-0.9% bis-acrylamide, sucrose (0%-10%) according to [45] using 8 cm glasses to cast gel. Samples (1 mL) were taken from reaction reactor using a 5 mL syringe at different times (0, 3, 6, 12, 24, 35 and 50 min) and reaction stopped by heating at 90°C for 10 min. Samples were centrifuged at 5,000 rpm for 20 minutes and 500 µL of MsFPH were concentrated 10-fold using speedvac system (Labconco Centrivap, USA). Five microliters of concentrated samples were loaded onto acrylamide gel prepared as mentioned above. Proteins were visualized following colloidal Coomassie brilliant blue G-250 [46].

2.6. Native R. glutini 18.4 source, identification and growth conditions

Yeast R. glutinis 18.4 was isolated from Paititi Natural Reserve (37°54′00′′ S, 057°49′00″ W) located at the southeast edge of La Peregrina mountain range, General Pueyrredón county, Buenos Aires province, Argentina. Biological material collection authorization was under DISPO-2021-528-GDEBA-DPFAAYRNMDAGP document (Provincia de Buenos Aires, Argentina). Protocol for isolation of yeast was performed as [47]. Preliminary isolation of yeast strains was performed using Wallerstein Laboratory medium agar plates [47]. Identification of red isolated strain was performed by sequencing of ribosomal ITS 5.8S [48] and compared to NCBI databank. R. glutinis 18.4 was growth in YPD medium (2% ^w/_v yeast extract, ^w/_v 1% peptone, 1% ^w/_v glucose) or in distilled water diluted MsFPH supplemented with 1% ^w/_v of glucose (MsFPH + D). Soluble protein concentration for MsFPH + D medium was set to ~ 3,6 mg/mL (equal content present in YPD), pH adjusted at 5.5 and autoclaved. Fresh colonies of yeast growing in solid YPD medium for 48 h at 28°C were used to inoculate 5 mL YPD medium and incubated overnight at 28°C with stirring (200 rpm). Cell concentration of precultures was adjusted to 2 x 10⁸ cells.mL^− 1 in sterile 0.85% ^w/_v NaCl. Subsequently, 1 mL of the preculture was used to inoculate 40 mL of fresh medium. Yeast cultures were incubated at 28°C with stirring at 200 rpm for 8 days. Samples were taking 1–2 times per day to quantified growth as cells.mL^− 1 using Neubauer chamber.

2.7. Carotene extraction, characterization and quantification

Carotenoid production analysis by R. glutinis 18.4 in YPD or MsFPH + D media was performed as [49]. Acetone extracts were dry under nitrogen flux and resuspended in 100 µL of HPLC grade absolute ethanol. With automatic injector Shimadzu® SIL 10AF, 10 µL of sample was injected using equal HPLC equipment previously described. Carotenoids were separated with same column and guard column previously described, which were maintained at 40°C in Shimadzu® CTO-10AS VP oven. An isocratic method was used with 60:40 Metanol (HPLC Grade): Ethanol (HPLC Grade) and the flow was 1 mL.min^− 1 using a Shimadzu® LC20AT pump. A standard curve was performed with pure β-carotene (Sigma Aldrich C9750) and carotenoid production was expressed relative to β-carotene. TLC separation of pigments extracted from R. glutinis 18.4 was carried out in 7:1 n-hexane : ethyl acetate solvent mix. Single colored spots were scraped off from TLC plates, pigments extracted form silica with acetone, dry and resuspended in HPLC grade absolute ethanol or again in acetone. Additionally, visible-UV spectrum from 400nm to 600nm of extracted compounds was perform using Shimadsu UV-1800 spectrophotometer.

2.8. Statistical analysis

Statistical analysis of data from growth curves and carotenoid production was carried out using R software [50]. Growth curves were analyzed by Biogrowth R package [51] and compared statistically with statmod package using compareGrowthCurve function (CGGC permutation test, [52],[53]) with 1000 permutations to estimate p-values. For carotenoid production analysis, data was transformed by application of cube root to HPLC quantified concentration in each treatment and time. Two-way ANOVA type III and Tukey with adjusted Bonferroni post-hoc was applied to determinate significant differences in carotenoid production between culture media and time.

3.1. Characterization of milled M. schmitti waste and MsFPH

Proximate composition analysis of milled waste and MsFPH was performed as an initial step to address the potential use of MsFPH as nutrient for R. glutinis growth (Table 1).

Table 1

Proximate composition analysis of *M. schmitti* waste and MsFPH.
	Ms waste	MsFPH
Moisture (%)	80,25 ± 0,19	91,89 ± 1,83
Ash (%)	2,80 ± 0,07	1,14 ± 0,20
Protein (%)	12,71 ± 0,81	5,71 ± 0,12
Non-protein nitrogen (%)	1,01 ± 0,05	0,36 ± 0,02
% = g/100g

Subsequently, we analyzed residual biomass of M. schmitti treated with Alcalase 2.4L. Treated biomass showed a ~ 45% of degree of hydrolysis (%DH) after 50 min (Fig. 1A) and additional time of reaction not generated impact in %DH (data not shown). SDS-PAGE analysis of samples taken at different times of hydrolysis shows a gradual increase in protein degradation with enrichment of ~ 10 kDa fraction of polypeptides at the end of treatment (Fig. 1B). Also, FAA enzymatically released over time was determinate. Kinetic of FAA liberation showed enrichment of all amino acids over time by 2–3 folds, except for tyrosine that reduce its concentration (Fig. 1C). The sum of individual concentration of each FAA in MsFPH was 7.27 ± 0.11 mg.mL^− 1.

3.2. Growth of native strain R. glutinis 18.4 using MsFPH as substitute nutrient source in growth media.

Bioprospection of native yeast from the southeast province of Buenos Aires, Argentina led to the isolation of 82 yeast-like strains with differential colony morphology in Wallerstein Laboratory medium agar plates (data not show). One of this isolated showing fast growth and bright red color was selected for molecular identification. This strain was identified as R. glutinis and named in this study R. glutinis 18.4.

Protein quantification by Lowry method indicate that MsFPH contains 16,48 ± 0,85 mg.mL^− 1 of soluble proteins while YPD medium (standard laboratory yeast growth medium) contains 3,63 ± 0,70 mg.mL^− 1 (Table 2). Additionally, we determinate total carbohydrates in MsFPH. As expected, a low value of total carbohydrate content was observed where MsFPH shows 5,30 ± 0,84 mM equivalents of glucose compared with 111 mM of glucose in YPD.

Table 2

Protein and FAA use by *R. glutinis*
	Protein (mg.mL^− 1)
	Initial	Final	% of use	Initial	Final	% of use
YPD	3,63 ± 0,70	3,64 ± 0,23	0	11,68 ± 0,01	7,99 ± 0,92	31,6
MsFPH + D	3,6 ± 0,00	2,39 ± 0,18	33,61	1,81 ± 0,02	0,60 ± 0,38	66,8

Results indicated that MsFPH has a ~ 4.5-fold higher concentration of proteins than YPD medium. According to this, to prepare MsFPH based medium, it was diluted with distilled water setting soluble protein concentration at 3,6 mg.mL^− 1 in both growth medium. Finally, supplementation with glucose 1% ^w/_v at a final concentration yields MsFPH + D growth medium. Important factor relevant to yeast growth was the concentration and profile of FAA in both MsFPH + D and YPD media. The FAA concentration in FPH based medium was 1,81 ± 0,02 mg.mL^− 1 (Table 2) while in YPD was 11,68 ± 0,01 mg.mL^− 1, and FAA profile differs extensively between both media (Fig. 2).

Growth curves of native R. glutinis 18.4 obtained in both medium show equal growth kinetics (Fig. 3, no statistical difference, p-value = 0,370) indicating that MsFPH + D contain all nutrients needed by yeast to grow and replace totally the use of peptone and yeast extract in YPD media. Comparison of initial and final concentration of proteins and FAA in both media (Table 2) indicate that R. glutinis growing on MsFPH + D medium use both proteins and FAA in more extensive way than in YPD. Also, in YPD medium yeast do not use proteins for growth.

As additional assay, we check if MsFPH alone (without added carbon source) could sustain growth of yeast (data not show). In some situations, microorganisms could use carbon bone of FAA and small oligopeptides as a carbon source [54]. In this experiment, R. glutinis 18.4 only reached 1x10⁸ cells.mL^− 1 (5 times lower than in YPD or MsFPH + D) indicating that the FAA and short oligopeptides could not compensate carbon absence.

3.3. Carotenoid production by R. glutinis 18.4 in MsFPH + D media

R. glutinis is a well characterized carotenogenic yeast [34]. The strain used in this study was identified at the molecular level as R. glutinis and it produces characteristic orange/red colonies on YPD plates (Fig. 4A). Preliminary isolation of pigments by TLC and UV-Visible analysis of acetone pigment extractions indicated the presence of β-carotene, torulene and torularhodin (Fig. 3B, C). Since native yeast could use MsFPH as nitrogen/nutrient source, we analyzed the reproduction of carotenoids in this new medium and compared to YPD.

Quantification by HPLC and analysis of individual peaks using photodiode array (PDA) equipment confirm the presence of β-carotene, torulene and torularhodin in R. glutinis 18.4. Production of each carotenoid was significatively different between YPD and MsFPH + D media (Fig. 5).

The concentration of β-carotene in YPD medium not change over the time of assay, while in MsFPH + D a gradual reduction was observed reaching 0,5 mg.L^− 1 at 142 hours of growth. For torulene, it concentration showed a complex dynamic in YPD showed a gradual increase until 116 hours of growth, lowered it concentration at the end of the experiment. On the other hand, in MsFPH + D concentrations were significantly higher compared to YPD at the beginning but dropped drastically in middle of experiment run and remained in this value until the end. For torularhodin pigment in YPD medium, the concentration of this carotenoid does not changed over time, while increases in MsFPH + D growth medium across experimental time. It is important to note that the torularhodin concentration at the end of experiment reach 129,54 ± 48,99 mg.L^− 1 in MsFPH + D medium while in YPD reached only 22,75 ± 10,22 mg.L^− 1, near to 6-fold increase in torularhodin production when MsFPH + D was used as the only source of nitrogen and nutrients. Differences in color of both cultures was appreciated by the naked eye (Fig. 5). The ratio of each carotenoid indicates that biosynthesis was directed to torularhodin production in both media, where for YPD the proportion was 9,99% β -carotene, 7,03% torulene and 82,97% torularhodin while in MsFPH + D was 0,71% β -carotene, 0,85% torulene and 98,43% torularhodin.

Fisheries and aquaculture production it is growing globally and accordingly waste generation follows the same trend. Since about two-thirds of the total amount of fish is considered waste, [2] strategies to valorize this biomass become key, not only from environmental but also from the economic point of view. Utilization of waste biomass to obtain bio-based products follow the biorefinery concept and contribute to more broad approach of circular economy [8].

The life cycle of M. schmitti constrains the sustainability of its fishery. Initiatives have emerged for the management and conservation of this species like to proposed by CTMFM [32]. Nevertheless, data indicate that it is extensively harvested both through artisanal and commercial activities, with a reported landing average of 2,862.1 metric tons between 2020 and 2023. In order to solve this situation, total biomass utilization of the already extracted individuals has been proposed, using the muscle to human feed and rest of biomass for recovery of lipid, proteins and others biomolecules encouraging maximal and sustainable use of M. schmitti fishing resources.

On the other hand, Rhodotorula sp. has been proposed as a platform for production of valuable biomolecules such as lipids, pigments and enzymes, making this yeast genus very promising for use as a microbial producer in an industrial scenario [55]. Rhodotorula sp. produce torulene and torularhodin carotenoids, among others. Both compounds have strong anti-oxidative and anti-microbial properties, and thus may be successfully used as food, feedstock, and cosmetics additives [35], [36]. There are currently a large number of Rhodotorula sp. strains considered to be of industrial interest [55]. However, the search for new wild strains may have advantages for industrial use, due to their robustness and the possibility of discovering new functions and/or metabolites of technological interest [56]. In this study, were used protein hydrolysates technology to obtain an aqueous fraction rich in proteins, oligopeptides, FAA and others nutrients from M. schmitti wastes, which was later used to generate microbial biomass of wild native R. glutinis that produces pigments with promising technological uses. Our focus for revalorization of biomass was pointed on nitrogen. Use of marine nitrogen for microbial biomass production in comparison to use chemical or plant/livestock nitrogen is very attractive. Marine nitrogen extracted from fish wastes initially comes from primary nitrogen fixation in the oceans [57]. On the contrary, chemical ammonium used on defined growth media is produced directly by Heber-Bosch reaction [58]. Even peptones from and animals sources ultimately and indirectly fall under the Heber-Bosch reaction, since the fertilizers used for plant growth as well as livestock feed were generated by this reaction.

As mentioned above, the production of FPH generates an aqueous solution containing oligopeptides and FAA. In addition to the presence of this nitrogen source in waste biomass, marine chondrichthyans are ion-regulating osmoconformers accumulating mainly urea osmolyte and in minor extent trimethylamine N-oxide (TMAO) [59]. Urea could be extracted in the aqueous fraction during hydrolysis and can serve as a nitrogen source for yeast [60]. In this regard, by analyzing the non-protein nitrogen data (NPN, Table 1) and relating it to the amount of FAA acids in MsFPH + D determined by HPLC, we can conclude that all NPN present in MsFPH + D medium corresponds to FAA quantified with apparent no presence of urea or other inorganic nitrogen form in MsFPH + D. This result is not surprising since waste biomass used for production of MsFPH + D medium was devoid of muscle and viscera. Although all tissues and blood of elasmobranchs contain urea as osmolyte, its concentration is focused on the kidney [61],[62] and muscle [59].

Carotenoid production by R. glutinis has been investigated in numerous studies employing defined media using inorganic nitrogen and carbon sources, peptone-based media, as well as in residual materials from various industries. Regarding the latter, studies have been conducted on carotenoid and lipid production using paddy straw hydrolyzate [63], waste glycerol and potato wastewater [64], olive mill wastewater [65], waste loquat kernels [66], waste chicken feathers [67] and using agro-industrial raw materials like grape must, glucose syrup, beet molasses, soybean flour extract and maize flour extract [68]. Our work is the first report of using fish proteins hydrolysate technology to growth carotenogenic yeast strain.

Comparison in torulene and torularhodin production (mg.L^− 1 of culture) with others reports [35] indicate a high level of production of strain 18.4. Recently [55] revised extensively growth conditions and reports the ratios between the different carotenoids produced by several R. glutinis strains. A comparison of our results with this data indicates that strain 18.4 using MsFPH + D medium has a higher proportion of torularhodin in comparison with others R. glutinis strains.

Production of torularhodin is higher in the MsFPH + D medium than in YPD at the end of cultivation time. The reason for this behavior it is unknown in our conditions, but despite that MsFPH + D support optimal yeast growth evaluated by cells.mL^− 1, this medium could generate some stressful conditions that generate accumulation of torularhodin. As shown, protein concentration in both culture media was set at same value but the FAA concentration in MsFPH + D is near 6-fold lower than YPD and could induce some nutritionally stressful conditions. In red yeast such as R. glutinis, torularhodin plays a crucial physiological role, primarily associated with its anti-oxidant and anti-bacterial properties under conditions of elevated oxidative stress [36]. Others aothors [69] found that under nitrogen starvation, Rhodosporidium toruloides (another carotenogenic red yeast) change transcriptomic expression of several enzymes involves in acetyl-CoA metabolism and deviated toward production of carotenoids. In addition, [70] found that glucose exhaustion was accompanied by the predominant formation of torularhodin in R. glutinis using intracellular oleic acid for biosynthesis. Another factor participating in the regulation of carotenoid biosynthesis regulation could be the carbon/nitrogen ratio. The use of varying concentrations of carbon and nitrogen sources, as well as the specific nature of either carbon or nitrogen source, represents an additional factor that influences carotenoid biosynthesis [70]. In this sense, both media (YPD and MsFPH + D) have the same carbon source at the same concentration, but differ not only in concentration of FAA, but also in relative quantities of each individual FAA, leading to differences in C/N ratios between both media. In addition, R. glutinis growing in MsFPH + D medium decrease protein concentration but not in YPD medium. This could indicate the activation of proteolytic activity trigger by nitrogen starvation in the MsFPH + D medium that does not occur in YPD [70]. YPD medium has a 6-fold higher concentration of FAA than MsFPH + D medium at the beginning of cultivation and compared with others similar works [72], FAA concentration in YPD even at the end of cultivation time was in sufficient quantities for optimal growth. On the other hand, in MsFPH + D medium final concentration of FAA and evidence of protein degradation indicate that nitrogen starvation could be present. Some of these factors among others could explain the differences observed in torularhodin production in R. glutinis growing in MsFPH + D medium and deserve more studies to dilucidated the mechanisms involved.

In summary, all together, these results show that from wastes of processing M. schmitti and using enzymatic hydrolysis technology, we can obtain oligopeptides, FAA and nutrients that support carotenogenic yeast growth. The generated biomass is further processed to obtain carotenoids pigments, important molecules with many technological applications. Next research steps involve understanding metabolism and/or genetic regulation of carotenoid synthesis driving by MsFPH in R. glutinis 18.4, valorization of residual yeast biomass obtained before carotenoid extraction (focus on protein recovery) and explore by-product synergy, with a focus on replace laboratory grade glucose by industrial by-products like glycerol from bioethanol or lactose from cheese production. Additionally, assays to increase production scale and test other fish by-products for FPH production will be perform.

Declaration of generative AI in scientific writing

Generative AI and AI-assisted technologies was used only in the writing process to improve the readability and language of the manuscript. After using AI tool, the corresponding author (AADP) reviewed and edited the content as needed and take full responsibility for the content of the published article.

Acknowledgment: To Esteban Zugasti owner of Paititi reserve for let us (AADP, CMP, CC) carry bioprospection project on her property, to Ing. Agr. Liz Echeverria for its invaluable help in the identification of native plant species under bioprospection project, to Martin and Rachel who provided scientific material for investigations, to Tito Paggi that provided β-carotene standard and INIDEP BIP Dr. Eduardo Holmberg crew who processed M. schmitti samples on board. This is an INIDEP contribution number xxxx.

Author contributor: Andres Arruebarrena Di Palma: conceptualization, methodology, investigation, formal analysis, resources and writing original draft and final manuscript. Yanina Turina: methodology and investigation on HPLC for amino acids and carotenoids determination. Rocio Isla Naveira: methodology for carotenoids extraction and UV-Vis characterization. Neonila Kulisz: investigation for proximate determinations. Cintia Mariana Pereyra: conceptualization, methodology, investigation and resources to isolation of native yeast in bioprospection project. Claudia Casalongué: resources and supervision on bioprospection project. Agueda Elena Massa: project administration to access of M. schmitti biomass samples, funding acquisition and supervision. All authors revised final manuscript.

Funding Sources: This work was supported by funds from INIDEP, CFP, CONICET (PIP 2021-2023), SPU (RESOL-2018-109-APN-SECPU#MECCYT) and ANPCyT, (PICT 2020 SERIE A –I-GRF (03019)).

Declaration of competing interest: The authors have no competing interests to declare that are relevant to the content of this article.

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The authors declare no competing interests.

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Fish waste valorization: protein hydrolysate as sustainable nitrogen and nutrients for production of carotenogenic yeasts biomass.

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Abstract

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Highlights

1. Introduction

2. Material and Methods

2.1. M. schmitti waste processing

2.2. Enzymatic hydrolysis

2.3. Proximate composition analysis

2.4. Measurement of soluble protein, carbohydrates and free amino acids

2.5. Gradient SDS-PAGE

2.6. Native R. glutini 18.4 source, identification and growth conditions

2.7. Carotene extraction, characterization and quantification

2.8. Statistical analysis

3. Results

3.1. Characterization of milled M. schmitti waste and MsFPH

3.3. Carotenoid production by R. glutinis 18.4 in MsFPH + D media

4. Discussion

Declarations

References

Additional Declarations

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