Reusing wastewater from Coffea arabica processing to produce single-cell protein using Candida sorboxylosa: Optimizing of culture conditions.

Coffee is a crop of significant socioeconomic importance, and the reuse of agri-food by-products and biowaste has great potential across several industries. Coffee wastewater (CWW) is a valuable resource containing essential nutrients that can be utilized by Candida sorboxylosa for single-cell protein (SCP) production. This utilization contributes to mitigating the negative impacts of agro-industrial waste. The optimization of culture conditions using the design of experiments (DoE) technique is crucial in understanding the environmental factors influencing metabolite production. In our study, the DoE technique was employed to analyze culture conditions, including room temperature, pH 8.4, agitation at 200 rpm, a headspace of 60% (v/v), and an inoculum of 0.75 DO600nm over 28-h period. This approach resulted in a remarkable SCP yield of 64.4% and dry cell weight (DCW) of 2.26 g/L. It is noteworthy that there is no literature reporting SCP production under alkaline pH conditions in yeast. Interestingly, our work demonstrated that an alkaline pH of 8.4 significantly influenced SCP production by C. sorboxylosa. The DoE technique proved to be an efficient statistical tool for optimizing culture conditions, offering several advantages, such as: (i) conducting cultures at room temperature to minimize unnecessary energy consumption; (ii) reducing the incubation time from 46 to 28 h, thereby enhancing overall productivity; (iii) achieving 1.7-fold increase in SCP yield compared to previous basal production levels.

Trends and prospects in dairy protein replacement in yogurt and cheese.

There is a growing demand for nutritional, functional, and eco-friendly dairy products, which has increased the need for research regarding alternative and sustainable protein sources. Plant-based, single-cell (SCP), and recombinant proteins are being explored as alternatives to dairy proteins. Plant-Based Proteins (PBPs) are commonly used to replace total dairy protein. However, PBPs are generally mixed with dairy proteins to improve their functional properties, which makes them dependent on animal protein sources. In contrast, single-Cell Proteins (SCPs) and recombinant dairy proteins are promising alternatives for dairy protein replacement since they provide nutritional components, essential amino acids, and high protein yield and can use industrial and agricultural waste as carbon sources. Although alternative protein sources offer numerous advantages over conventional dairy proteins, several technical and sensory challenges must be addressed to fully incorporate them into cheese and yogurt products. Future research can focus on improving the functional and sensory properties of alternative protein sources and developing new processing technologies to optimize their use in dairy products. This review highlights the current status of alternative dairy proteins in cheese and yogurt, their functional properties, and the challenges of their use in these products.

Microbial meat: A sustainable vegan protein source produced from agri-waste to feed the world.

In the modern world, animal and plant protein may not meet the sustainability criteria due to their high need for arable land and potable water consumption, among other practices. Considering the growing population and food shortage, finding alternative protein sources for human consumption is an urgent issue that needs to be solved, especially in developing countries. In this context, microbial bioconversion of valuable materials in nutritious microbial cells represent a sustainable alternative to the food chain. Microbial protein, also known as single-cell protein (SCP), consist of algae biomass, fungi or bacteria that are currently used as food source for both humans and animals. Besides contributing as a sustainable source of protein to feed the world, producing SCP, is important to reduce waste disposal problems and production costs meeting the sustainable development goals. However, for microbial protein as feed or food to become an important and sustainable alternative, addressing the challenges of raising awareness and achieving wider public regulatory acceptance is real and must be addressed with care and convenience. In this work, we critically reviewed the potential technologies for microbial protein production, its benefits, safety, and limitations associated with its uses, and perspectives for broader large-scale implementation. We argue that the information documented in this manuscript will assist in developing microbial meat as a major protein source for the vegan world.

Growth of Methylobacterium organophilum in Methanol for the Simultaneous Production of Single-Cell Protein and Metabolites of Interest.

Research background: This study aims to monitor the growth of the methylotrophic bacteria Methylobacterium organophilum in a culture medium with methanol as a carbon source and to verify the production of unicellular proteins and other biomolecules, such as carotenoids, exopolysaccharides and polyhydroxyalkanoates, making them more attractive as animal feed. Experimental approach: Bacterial growth was studied in shake flasks using different carbon/nitrogen (C:N) ratios to determine their best ratio for achieving the highest volumetric productivity of cells and substrate consumption rate. This optimal parameter was further used in a fed-batch operating bioreactor system to define the kinetic profile of cell growth. Methanol consumption was measured by HPLC analysis and the extracted pigments were analyzed by liquid chromatography/mass spectrometry. Chemical composition and rheological properties of the produced exopolysaccharides were also determined. Results and conclusions: The best experimental parameters were verified using an initial methanol concentration of 7 g/L in the culture medium. The same initial substrate concentration was used in the fed-batch operation and after 60 h of cultivation 5 g/L of biomass were obtained. The accumulation of carotenoids associated with cell growth was monitored, reaching a concentration of 1.6 mg/L at the end of the process. These pigments were then analyzed and characterized as a set of xanthophylls (oxidized carotenoids). In addition, two other product types were identified during the fed-batch operation: exopolysaccharides, which reached a concentration of 8.9 g/L at the end of the cultivation, and an intracellular granular structure that was detected by transmission electron microscopy (TEM), suggesting the accumulation of polyhydroxyalkanoate (PHA), most likely polyhydroxybutyrate. Novelty and scientific contribution: Methylobacterium organophilum demonstrated a unique ability to produce compounds of commercial interest. The distinct metabolic diversity of this bacterium makes room for its use in biorefineries.

Prospective production of fructose and single cell protein from date palm waste

BACKGROUND: Fructose and single cell protein are important products for the food market. Abundant amounts of low-grade dates worldwide are annually wasted. In this study, highly concentrated fructose syrups and single cell protein were obtained through selective fermentation of date extracts by Saccharomyces cerevisiae. RESULTS: The effect of air flow (0.1, 0.5, 0.75, 1, 1.25 and 1.5 vvm) and pH (4.5, 4.8, 5, 5.3 and 5.6) was investigated. Higher air flow led to lower fructose yield. The optimum cell mass production of 10 g/L was achieved at air flow of 1.25 vvm with the fructose yield of 91%. Similar cell mass production was obtained in the range pH of 5.0­5.6, while less cell mass was obtained at pH less than 5. Controlling the pH at 4.5, 5.0 and 5.3 failed to improve the production of cell mass which were 5.6, 5.9 and 5.4 g/L respectively; however, better fructose yield was obtained. CONCLUSIONS: Extension of the modified Gompertz enabled excellent predictions of the cell mass, fructose production and fructose fraction. The proposed model was also successfully validated against data from literatures. Thus, the model will be useful for wide application of biological processes.

Influencing factors on single-cell protein production by submerged fermentation: a review

Since more than twenty years ago, some species of bacteria and fungi have been used to produce protein biomass or single-cell protein (SCP), with inexpensive feedstock and wastes being used as their sources of carbon and energy. The role of SCP as a safe food and feed is being highlighted more because of the worldwide protein scarcity. Even though SCP has been successfully commercialized in the UK for decades, study of optimal fermentation conditions, various potential substrates, and a broad range of microorganisms is still being pursued by many researchers. In this article, commonly used methods for the production of SCP and different fermentation systems are briefly reviewed, with submerged fermentation being highlighted as a more commonly used method. Emphasis is given to the effect of influencing factors on the biomass yield and productivity in an effort to provide a comprehensive review for researchers in related fields of interest.

Multiscale design of a dairy beverage model composed of Candida utilis single cell protein supplemented with oleic acid.

One of the main challenges in the food industry is to design strategies for the successful incorporation of natural sources of bioactive compounds. Recently, yogurts and other fermented dairy beverages have been proposed as ideal carriers of such bioactive compounds such as fatty acids and antioxidants that could improve consumers' health. However, the incorporation of new ingredients causes functional and structural modifications that may affect the consumers' preferences. In this work, a dairy beverage model supplemented with oleic acid has been designed by partial substitution of milk by Candida utilis single-cell protein extract. The changes in the structural properties of this new beverage were evaluated by following the fermentation process, pH, aggregate size, microstructure, and changes in rheological properties. Furthermore, molecular dynamics simulations were carried out to analyze the interaction between its main components. Our data revealed that samples with a percentage of milk substitution of 30% showed a higher viscosity as compared with the other percentages and less viscosity than the control (no substitution). These samples were then selected for fortification by incorporating oleic acid microcapsules. A concentration of 1.5 g/100 g was shown to be the optimal quantity of microcapsules for oleic acid supplementation. Molecular dynamic simulations revealed glutathione as an important component of the micro-gel structure. The present study forms the basis for novel studies where Candida utilis single-cell protein and microencapsulated essential oils could be used to design innovative bioproducts.

Effect of initial pH of medium with potato wastewater and glycerol on protein, lipid and carotenoid biosynthesis by Rhodotorula glutinis

Background: Rhodotorula glutinis is capable of synthesizing numerous valuable metabolites with extensive potential industrial usage. This paper reports the effect of initial culture medium pH on growth and protein, lipid, and carotenoid biosynthesis by R. glutinis. Results: The highest biomass yield was obtained in media with pH 4.0­7.0, and the value after 72 h was 17.2­19.4 gd.w./L. An initial pH of the medium in the range of 4.0­7.0 has no significant effect on the protein (38.5­41.3 g/100 gd.w.), lipid (10.2­12.7 g/100 gd.w.), or carotenoid (191.7­202.9 µg/gd.w.) content in the biomass or on the profile of synthesized fatty acids and carotenoids. The whole pool of fatty acids was dominated by oleic (48.1­53.4%), linoleic (21.4­25.1%), and palmitic acids (13.0­15.8%). In these conditions, the yeast mainly synthesized torulene (43.5­47.7%) and ß-carotene (34.7­38.6%), whereas the contribution of torularhodin was only 12.1­16.8%. Cultivation in medium with initial pH 3.0 resulted in a reduction in growth (13.0 gd.w./L) and total carotenoid (115.8 µg/gd.w.), linoleic acid (11.5%), and torularhodin (4.5%) biosynthesis. Conclusion: The different values of initial pH of the culture medium with glycerol and deproteinized potato wastewater had a significant effect on the growth and protein, lipid, and carotenoid biosynthesis by R. glutinis.

Potential application of microalga Spirulina platensis as a protein source.

The high protein level of various microalgal species is one of the main reasons to consider them an unconventional source of this compound. Spirulina platensis stands out for being one of the richest protein sources of microbial origin (460-630 g kg-1 , dry matter basis), having similar protein levels when compared to meat and soybeans. The use of S. platensis in food can bring benefits to human health owing to its chemical composition, since it has high levels of vitamins, minerals, phenolics, essential fatty acids, amino acids and pigments. Furthermore, the development of new protein sources to supply the shortage of this nutrient is an urgent need, and protein from S. platensis plays an important role in this scenario. In this sense, extraction processes that allow maximum protein yield and total utilization of biomass is an urgent need, and ultrasonic waves have proven to be an effective extraction technique. The number of scientific papers related to protein fraction from S. platensis is still limited; thus further studies on its functional and technological properties are needed. © 2016 Society of Chemical Industry.

Mixed yeasts inocula for simultaneous production of SCP and treatment of vinasse to reduce soil and fresh water pollution.

This study evaluated the use of vinasse as a substrate for microbial biomass production and its disposal impact on the environment. After grown in vinasse, the microbial biomass (SCP) of two Saccharomyces cerevisiae strains, CCMA 0137 and CCMA 0188, showed high levels of essential amino acids (3.78%), varying levels of chemical elements, and low nucleic acid content (2.38%), i. e, good characteristics to food supplemements. Following biological treatment, spent vinasse biochemical oxygen demand (BOD) and chemical oxygen demand (COD) decreased to 51.56 and 29.29%, respectively. Cultivation with S. cerevisiae significantly reduced short term phytotoxicity and toxicity on soil microbiota of spent vinasse.

Protein enrichment of brewery spent grain from Rhizopus oligosporus by solid-state fermentation.

Brewery spent grain represents approximately 85 % of total by-products generated in a brewery. Consisting of carbohydrates, fiber, minerals and low amounts of protein, the use of brewery spent grain is limited to the feeding of ruminants; however, its potential use should be investigated. The reuse of this by-product using microorganisms by solid-state fermentation process as the case of protein enrichment by single-cell protein incorporation is an alternative to ensure sustainability and generate commercially interesting products. In this context, the aim of this study was to grow Rhizopus oligosporus in brewery spent grain under different initial moisture contents and nitrogen sources to increase the protein content of the fermented material. After 7 days of fermentation, increase of 2-4 times in the crude protein and soluble protein content was verified, respectively, compared to unfermented brewery spent grain. The kinetics of protein enrichment demonstrated the possibility of application of this technique, which can be a great alternative for use in diets for animals.

Evaluation of the composition of culture medium for yeast biomass production using raw glycerol from biodiesel synthesis

The work herewith investigated the production of yeast biomass as a source of protein, using Yarrowia lipolytica NRRL YB-423 and raw glycerol from biodiesel synthesis as the main carbon source. A significant influence of glycerol concentration, initial pH and yeast extract concentration on biomass and protein content was observed according to the 2v5-1 fractional design. These factors were further evaluated using a central composite design and response surface methodology, and an empirical model for protein content was established and validated. The biomass of Yarrowia lipolytica NRRL YB-423 reached 19.5 ± 1.0 g/L in shaken flasks cultivation, with a protein content of 20.1 ± 0.6% (w/w).

Evaluation of the composition of culture medium for yeast biomass production using raw glycerol from biodiesel synthesis.

The work herewith investigated the production of yeast biomass as a source of protein, using Yarrowia lipolytica NRRL YB-423 and raw glycerol from biodiesel synthesis as the main carbon source. A significant influence of glycerol concentration, initial pH and yeast extract concentration on biomass and protein content was observed according to the 2v (5-1) fractional design. These factors were further evaluated using a central composite design and response surface methodology, and an empirical model for protein content was established and validated. The biomass of Yarrowia lipolytica NRRL YB-423 reached 19.5 ± 1.0 g/L in shaken flasks cultivation, with a protein content of 20.1 ± 0.6% (w/w).

Evaluation of the composition of culture medium for yeast biomass production using raw glycerol from biodiesel synthesis

The work herewith investigated the production of yeast biomass as a source of protein, using Yarrowia lipolytica NRRL YB-423 and raw glycerol from biodiesel synthesis as the main carbon source. A significant influence of glycerol concentration, initial pH and yeast extract concentration on biomass and protein content was observed according to the 2v5-1 fractional design. These factors were further evaluated using a central composite design and response surface methodology, and an empirical model for protein content was established and validated. The biomass of Yarrowia lipolytica NRRL YB-423 reached 19.5 ± 1.0 g/L in shaken flasks cultivation, with a protein content of 20.1 ± 0.6% (w/w).

Producción de biomasa de Saccharomyces cerevisiae y Candida utilis usando residuos de pulpa de Coffea arabica L

Los avances en biotecnología industrial ofrecen oportunidades potenciales para la utilización económica de residuos agro-industriales tales como la pulpa de café, material mucilaginoso, fibroso (producto secundario) obtenido durante el proceso húmedo o seco del beneficio de las cerezas de café. El propósito de este trabajo fue utilizar los residuos de la pulpa de café, rico en materia orgánica, como sustrato para la producción de biomasa de levaduras por procesos de fermentación aeróbica. Los residuos de café se sometieron a hidrólisis con una solución de ácido sulfúrico al 2%, en una relación 10:1 (líquido:sólido), con un tamaño de partícula ≤ 2 mm., operando a presión atmosférica, ebullición a reflujo, durante 4 horas. El extracto ácido se filtró y se ajustó a pH 4,5 y luego se esterilizó a 120 ºC por 15 minutos. La fermentación se realizó con Saccharomyces cerevisiae y Candida utilis, medio de producción extracto de café enriquecido con sales nutritivas. Se formularon diferentes medios de producción (1,2,3 y 4), siendo el N°3, enriquecido con extracto de café hidrolizado, 1L; urea, 3g/L; fosfato ácido de potasio, 2g/L; extracto de malta, 1,3g/L y melaza, 30g/L, el cual aportó los mejores resultados. El tiempo total de fermentación fue de 8 horas. Se obtuvo 10g/L de biomasa con un incremento proteico de 7,39 a 42,5%. Se puede concluir que la pulpa de café constituye un sustrato adecuado para obtener biomasa o proteína unicelular, que podría ser destinada como suplemento en formulaciones para alimentación animal.

The advances in industrial biotechnology offer potential opportunities for the economic use of agro-industrial remainders such as the coffee pulp, mucilagenous, fibrous material (secondary product) obtained during the humid or dry process of the benefit of the coffee cherries. The intention of this work was to use the remainders of the pulp of coffee, rich in organic matter, like substrate for the production of biomass of leavenings by processes of aerobic fermentation. The coffee remainders were put under hydrolysis with a sulfuric acid solution to 2%, in a 10:1 relation (liquid: solid), with a size of particle ≤ 2 mm, operating to atmospheric pressure, boiling to ebb tide, during 4 hours. The acid extract filtered and it adjusted to pH 4,5 and then it sterilize to 120 ºC by 15 minutes. The fermentation was made with Saccharomyces cerevisiae and Candida utilis, the production means: extract of coffee enriched with nutritious salts. Different means from production were formulated (1,2,3 and 4), being the N°3, enriched with extract of hydrolyzed coffee, 1L; urea, 3g/L; acid potassium phosphate, 2g/L; extract of Malta, 1,3g/L and molasses, 30g/L, which contributed the best results. The total time of fermentation was of 8 hours. 10g/L of biomass with a protein increase from 7.39 to 42.5% was obtained. It is possible to be concluded that the coffee pulp constitutes an suitable substrate to obtain biomass or unicellular protein, that could be destined like supplement in formulations for feeding animal.

Industrial derivative of tallow: a promising renewable substrate for microbial lipid, single-cell protein and lipase production by Yarrowia lipolytica

The aim of the present study was to assess the potential of valorisation of a solid industrial derivative of tallow, composed of saturated free-fatty acids ("stearin"), by fermentations carried out by the yeast Yarrowia lipolytica ACA-DC 50109 in order to produce microbial lipid, biomass and extra-cellular lipase. High quantities of biomass were produced (biomass yield of around 1.1 ± 0.1 g of total biomass produced per g of fat consumed) when the organism was grown in shake flasks, regardless of the concentration of extra-cellular nitrogen present. Cellular lipids accumulated in notable quantities regardless of the nitrogen availability of the medium, though this process was clearly favoured at high initial fat and low initial nitrogen concentrations. The maximum quantity of fat produced was 7.9 mg/ml corresponding to 52.0% (wt/wt) of lipid in the dry biomass. Lipase production was critically affected by the medium composition and its concentration clearly increased with increasing concentrations of fat and extra-cellular nitrogen concentration reaching a maximum level of 2.50 IU/ml. Lipase concentration decreased in the stationary growth phase. In bioreactor trials, in which higher agitation and aeration conditions were employed compared with the equivalent trial in the flasks, significantly higher quantities of biomass were produced (maximum concentration 30.5 mg/ml, yield of 1.6 g of total biomass produced per g of fat consumed) while remarkably lower quantities of cellular lipids and extra-cellular lipase were synthesised. Numerical models successfully simulated both conversion of substrate fat into biomass and production and subsequent hydrolysis of extra-cellular lipase and presented a satisfactory predictive ability verifying the potential for single-cell protein and lipase production by Yarrowia lipolytica ACA-DC 50109. In all cultures, the mycelial form of the culture was dominant with few single cells present.

Effect of single cell protein as a protein source in Drosophila culture

The effects of biomass of Phanerochaete chrysosporium at various concentrations (1; 2.5; 5; 10; 25; 50; 75 and 100%) on the growth of Drosophila melanogaster have been investigated. Biomass was used as a protein source instead of corn flour in Standard Drosophila Medium(SDM). It was seen that it causes increments in both body size of larvae and the numbers of offsprings, especially at higher concentrations (50, 75 and 100%). At the application of 100% concentration, metamorphosis accelerated and was completed one day before the control. There were phenotipic abnormalities in both control (0.64%) and applications with low concentrations (0.02-0.19%), while they were not observed at the application of 100% concentration

Investigou-se os efeitos de biomassa de Phanerochaete chrysosporium em diferentes concentrações (1; 2,5; 5; 10; 25; 50; 75 e 100%) no crescimento de Drosophila melanogaster. Em meio SDM (Standard Drosophila Medium) a biomassa foi empregada como fonte de proteína, em substituição à farinha de milho. Observou-se que a biomassa causou aumento no tamanho corporal das larvas e no número de descendentes, especialmente nas concentrações mais elevadas (50, 75 e 100%). Ao usar-se a concentração de 100%, a metamorfose acelerou-se e completou-se um dia antes do controle. Foram observadas anormalidades fenotípicas tanto no meio controle (0,64%) quanto no meio contendo baixas concentrações de biomassa (0,02 a 0,19%), mas essas anormalidades não foram observadas na concentração de 100%.