Filamentous fungi for sustainable vegan food production systems within a circular economy: Present status and future prospects.

Filamentous fungi serve as potential candidates in the production of different value-added products. In the context of food, there are several advantages of using filamentous fungi for food. Among the main advantages is that the fungal biomass used food not only meets basic nutritional requirements but that it is also rich in protein, low in fat, and free of cholesterol. This speaks to the potential of filamentous fungi in the production of food that can substitute animal-derived protein sources such as meat. Moreover, life-cycle analyses and techno-economic analyses reveal that fungal proteins perform better than animal-derived proteins in terms of land use efficiency as well as global warming. The present article provides an overview of the potential of filamentous fungi as a source of food and food supplements. The commercialization potential as well as social, legal and safety issues of fungi-based food products are discussed.

Development of Meat Substitutes from Filamentous Fungi Cultivated on Residual Water of Tempeh Factories.

In recent years, there has been an increased motivation to reduce meat consumption globally due to environmental and health concerns, which has driven the development of meat substitutes. Filamentous fungal biomass, commonly known as mycoprotein, is a potential meat substitute since it is nutritious and has filaments to mimic meat fibrils. The current study aimed to investigate the potential use of a cheap substrate derived from the food industry, i.e., residual water in a tempeh factory, for mycoprotein production. The type of residual water, nutrient supplementation, optimum conditions for biomass production, and characteristics of the mycoprotein were determined. The results showed that the residual water from the first boiling with yeast extract addition gave the highest mycoprotein content. The optimum growth condition was a pH of 4.5 and agitation of 125 rpm, and it resulted in 7.76 g/L biomass. The mycoprotein contains 19.44% (w/w) protein with a high crude fiber content of 8.51% (w/w) and a low fat content of 1.56% (w/w). In addition, the amino acid and fatty acid contents are dominated by glutamic acid and polyunsaturated fatty acids, which are associated with an umami taste and are considered healthier foods. The current work reveals that the residual boiling water from the tempeh factory can be used to produce high-quality mycoprotein.

Correlation between the chemical, microbiological and sensory characteristics of cream cheese using a mixed and single probiotic culture.

The use of mixed culture in the fermentation industry requires more complicated equipment, processes, and monitoring systems; therefore, a single culture may be preferable. This study aimed to investigate the correlation between chemical and microbiological properties and sensory characteristics. In addition, this study aimed to determine the different characteristics of cheese made using single probiotic cultures of Lactobacillus plantarum Dad-13 and Lactobacillus plantarum Kita-3 and mixed culture. The obtained results showed that the chemical characteristics of cream cheese made using single cultures were similar to those of cream cheese made using mixed cultures. The viability of the cells remained high after 35 days of storage for the single culture, which was not the case for the mixed culture. In terms of sensory analysis, cream cheese made using the single culture L. plantarum Kita-3 showed higher overall liking score among the samples, which might correlate with the high ester and ketone content. In addition, there was a strong relationship between the ethyl octanoate and methyl butanol contents and the overall liking score. The results of this study showed that the use of a single culture of L. plantarum Kita-3 could improve the sensory characteristics of cream cheese with probiotic properties. This study also contributed to the development of cream cheese production, particularly in the screening of potential starters.

Study of Viability, Storage Stability, and Shelf Life of Probiotic Instant Coffee Lactiplantibacillus plantarum Subsp. plantarum Dad-13 in Vacuum and Nonvacuum Packaging at Different Storage Temperatures.

Probiotic coffee is an alternative to processed coffee that is preferred and can improve the balance of intestinal microflora so that it has a positive impact on health. Cell viability of probiotics may decrease during storage. Factors that can affect viability during storage are storage temperature, packaging, oxygen, and water activity. This study is aimed at evaluating the viability, storage stability, and shelf life of the probiotic instant coffee Lactiplantibacillus plantarum subsp. plantarum Dad-13 in vacuum and nonvacuum aluminium foil packaging and different storage temperatures. This study used a complete randomised design with three replicates of treatments. They were packaged using 90 µm thick aluminium foil in a vacuum and nonvacuum and stored at 4°C and 30°C for 50 days and 37°C for 15 days. Based on the literature, a temperature of 4°C can maintain the viability of probiotics for more than one month, the temperature commonly used to store dry products is room temperature (30°C), so longer storage (50 days) is tried. Meanwhile, to accelerate the prediction of quality degradation, extreme temperatures were used based on the literature that the viability of probiotics decreased drastically after being stored at 37°C for 7 days, then tried for longer storage (15 days). The evaluation of product was carried by sensory testing by comparing commercial instant coffee. The product has been tested for cell viability, water activity, and shelf life. The result showed that the colour attribute was significantly different for all formulations. The bitterness of probiotic instant coffee differed significantly from other formulations. The commercial instant coffee was preferred by panellist in terms of colour and bitterness. The aroma, sweetness, and overall attributes of all formulations were not significantly different. The cell viability in vacuum was higher than nonvacuum treatment, and it was higher in 4°C. However, cell viability for all treatments and during storage was still above 107 log CFU/g. Water activity in probiotic instant coffee with vacuum packaging is lower than in nonvacuum and stored at 4°C lower than in other temperatures. However, all treatments were still below 0.60. The shelf life of products reaches two years when they are stored in vacuum packaging at 4°C while a temperature of 30°C reaches 3 months. So, the panellists accepted probiotic instant coffee, vacuum packaging, and low temperature could maintain viability, stability, and longer shelf life.

Household fermentation of leftover bread to nutritious food.

Resource dependency of food production is aggravated when food is wasted. In Sweden, it is estimated that 37% of the total bread waste is generated at the household level. This work aimed to assess whether fermentation using edible filamentous fungi at households can provide a solution to valorize leftover bread in the production of fungi-based food for consumption. Bread was fermented in household and laboratory conditions with Neurospora intermedia and Rhizopus oligosporus. The results show that bread can be successfully and easily fermented at households, without signs of microbial contamination even though the conditions were not sterile. Fermentation at the household resulted in higher protein, fat and fiber content as well as greater starch reduction compared to the samples fermented under laboratory conditions. Household engagement in bread fermentation will likely depend on values that motivate reusing leftover bread. Perceived values that are expected to motivate engagement vary across individuals, but may include improved nutritional benefits, food waste prevention, convenience, responsibilities, and being part of sustainable societies and actions.

Protective effect of a reverse membrane bioreactor against toluene and naphthalene in anaerobic digestion.

Raw syngas contains tar contaminants including toluene and naphthalene, which inhibit its conversion to methane. Cell encasement in a hydrophilic reverse membrane bioreactor (RMBR) could protect the cells from hydrophobic contaminants. This study aimed to investigate the inhibition of toluene and naphthalene and the effect of using RMBR. In this work, toluene and naphthalene were added at concentrations of 0.5-1.0 and 0.1-0.2 g/L in batch operation. In continuous operation, concentration of 0-6.44 g/L for toluene and 0-1.28 g/L for naphthalene were studied. The results showed that no inhibition was observed in batch operation for toluene and naphthalene at concentrations up to 1 and 0.2 g/L, respectively. In continuous operation of free cell bioreactors (FCBRs), inhibition of toluene and naphthalene started at 2.05 and 0.63 g/L, respectively. When they were present simultaneously, inhibition of toluene and naphthalene occurred at concentrations of 3.14 and 0.63 g/L, respectively. In continuous RMBRs, no inhibition for toluene and less inhibition for naphthalene were observed, resulting in higher methane production from RMBR than that of FCBR. These results indicated that RMBR system gave a better protection effect against inhibitors compared with FCBR.

The role of filamentous fungi in advancing the development of a sustainable circular bioeconomy.

Human activities generate enormous amounts of organic wastes and residues. Filamentous fungi (FF) are able to grow on a broad range of substrates and survive over a wide spectrum of growth conditions. These characteristics enable FF to be exploited in biorefineries for various waste streams. Valorization of food industry byproducts into biomass and various arrays of value-added products using FF creates promising pathways toward a sustainable circular economy. This approach might also contribute to reaching the sustainable development goals set by the United Nations, particularly for zero hunger as well as affordable and clean energy. This paper presents the application of filamentous fungi in food, feeds, fuels, biochemicals, and biopolymers. The nutritional values, health benefits, and safety of foods derived from byproducts of food industries are also addressed. The technoeconomical feasibilities, sustainability aspects and challenges and future perspectives for biorefineries using filamentous fungi are discussed.

Application of cell culture technology and genetic engineering for production of future foods and crop improvement to strengthen food security.

The growing population and the climate changes put a pressure on food production globally, therefore a fundamental transformation of food production is required. One approach to accelerate food production is application of modern biotechnology such as cell culture, marker assisted selection, and genetic engineering. Cell culture technology reduces the usage of arable land, while marker-assisted selection increases the genetic gain of crop breeding and genetic engineering enable to introduce a desired traits to crop. The cell culture technology has resulted in development of cultured meat, fungal biomass food (mycoprotein), and bioactive compounds from plant cell culture. Except cultured meat which recently begin to penetrate the market, the other products have been in the market for years. The marker-assisted selection and genetic engineering have contributed significantly to increase the resiliency against emerging pests and abiotic stresses. This review addresses diverse techniques of cell culture technology as well as advanced genetic engineering technology CRISPR Cas-9 and its application for crop improvement. The pros and cons of different techniques as well as the challenges and future perspective of application of modern biotechnology for strengthening food security are also discussed.

Adhesion Properties of Lactobacillus plantarum Dad-13 and Lactobacillus plantarum Mut-7 on Sprague Dawley Rat Intestine.

Adhesion capacity is considered one of the selection criteria for probiotic strains. The purpose of this study was to determine the adhesion properties of two candidate probiotics, Lactobacillus plantarum Dad-13 and Lactobacillus plantarum Mut-7. The evaluation included the hydrophobicity of the cell surface using microbial adhesion to hydrocarbons (MATH), autoaggregation, and the adhesion of L. plantarum Dad-13 and L. plantarum Mut-7 to the intestinal mucosa of Sprague Dawley rat, followed by genomic analysis of the two L. plantarum strains. L. plantarum Dad-13 and L. plantarum Mut-7 showed a high surface hydrophobicity (78.9% and 83.5%) and medium autoaggregation ability (40.9% and 57.5%, respectively). The exposure of both isolates to the surface of the rat intestine increased the total number of lactic acid bacteria on the colon compartment, from 2.9 log CFU/cm2 to 4.4 log CFU/cm2 in L. plantarum Dad-13 treatment and to 3.86 log CFU/cm2 in L. plantarum Mut-7 treatment. The results indicate the ability of two L. plantarum to attach to the surface of the rat intestine. The number of indigenous E. coli in the colon also decreased when the compartment was exposed to L. plantarum Dad-13 and Mut-7, from 2.9 log CFU/cm2 to 1 log CFU/cm2. Genomic analysis revealed that both strains have genes related to adhesion properties that could play an important role in increasing the adherence of probiotics to the intestinal mucosa such as gene encoding fibronectin-binding protein, chaperonin heat shock protein 33 (Hsp33), and genes related to the capsule and cell wall biosynthesis. Based on these findings, we believe that L. plantarum Dad-13 and L. plantarum Mut-7 have adhesion properties to the intestinal mucosa in the rat intestine model system. The present research will be essential to elucidate the molecular mechanism associated with adhesion in our two probiotic strains.

Cultivation of edible filamentous fungus Aspergillus oryzae on volatile fatty acids derived from anaerobic digestion of food waste and cow manure.

In a circular economy approach, edible filamentous fungi (single cell protein) can be cultivated on volatile fatty acids (VFAs) derived from anaerobic digestion (AD) of organic-rich waste streams. In this study, the effect of pH, concentration/distribution of VFAs, nutrient supplementation, and type of waste on Aspergillus oryzae cultivation on synthetic VFAs, and actual VFAs derived from AD of food waste and cow manure were investigated. The optimal pH for A. oryzae growth on VFAs were 6 and 7 with maximum acetic acid consumption rates of 0.09 g/L.h. The fungus could thrive on high concentrations of acetic (up to 9 g/L) yielding 0.29 g dry biomass/gVFAsfed. In mixed VFAs cultures, A. oryzae primarily consumed caproic and acetic acids reaching a biomass yield of 0.26 g dry biomass/gVFAsfed (containing up to 41% protein). For waste-derived VFAs at pH 6, the fungus successfully consumed 81-100% of caproic, acetic, and butyric acids.

Enhancing or Inhibitory Effect of Fruit or Vegetable Bioactive Compound on Aspergillus niger and A. oryzae.

Fruit and vegetable processing wastes are global challenges but also suitable sources with a variety of nutrients for different fermentative products using bacteria, yeast or fungi. The interaction of microorganisms with bioactive compounds in fruit waste can have inhibitory or enhancing effect on microbial growth. In this study, the antimicrobial effect of 10 bioactive compounds, including octanol, ellagic acid, (-)-epicatechin, quercetin, betanin, ascorbic acid, limonene, hexanal, car-3-ene, and myrcene in the range of 0-240 mg/L on filamentous fungi Aspergillus oryzae and Aspergillus niger were investigated. These fungi were both found to be resistant to all compounds except octanol, which can be used as a natural antifungal agent, specifically against A. oryzae and A. niger contamination. On the contrary, polyphenols (quercetin and ellagic acid), ascorbic acid, and hexanal enhanced A. niger biomass yield 28%, 7.8%, 16%, and 6%, respectively. Furthermore, 240 mg/L car-3-ene was found to increase A. oryzae biomass yield 8%, while a 9% decrease was observed at lower concentration, 24 mg/L. Similarly, up to 17% decrease of biomass yield was observed from betanin and myrcene. The resistant nature of the fungi against FPW bioactive compounds shows the potential of these fungi for further application in waste valorization.

Utilization of food waste-derived volatile fatty acids for production of edible Rhizopus oligosporus fungal biomass.

Rhizopus oligosporus is an edible filamentous fungus that can contribute to meet the growing demand for single-cell protein. Volatile fatty acids (VFAs) are favorable potential substrates for producing R. oligosporus biomass due to their capacity to be synthesized from a wide range of low-value organic solid wastes via anaerobic digestion. The goal of this work was to cultivate R. oligosporus using food waste-derived VFAs as the sole carbon source. To maintain the requisite low substrate concentrations, the fed-batch cultivation technique was applied. This resulted in a four-fold improvement in biomass production relative to standard batch cultivation. Maximum biomass yield of 0.21 ± 0.01 g dry biomass/g VFAs COD eq. consumed, containing 39.28 ± 1.54% crude protein, was obtained. In the bubble-column bioreactors, the complete uptake of acetic acid was observed, while the consumptions of caproic and butyric acids reached up to 97.64% and 26.13%, respectively.

Assessment of Microbiological Quality and Mycotoxin in Dried Chili by Morphological Identification, Molecular Detection, and Chromatography Analysis.

The growing interest in spicy foods leads to the global demand for spices, particularly dried chili. This study aimed to assay both aflatoxin (AFs) and ochratoxin A (OTA) contamination using an integrative method of morphological identification, molecular detection, and chromatography analysis on dried chili provided from traditional and modern markets in Indonesia. The results showed that total fungal infection ranged from 1-408 × 103 CFU/g. Eighty percent of the chili obtained from both the traditional and the modern markets were infected by Aspergillus spp., in which 50% of the infections were identified as A. parasiticus and A. flavus. A complete set of targeted genes involved in AF production and OTA were detected in two isolates of A. flavus and one isolate of A. carbonarius, respectively. The levels of AFs B1, B2, and OTA in the contaminated dried chilies were in the range of 39.3-139.5 µg/kg, 2.6-33.3 µg/kg, and 23.7-84.6 µg/kg, respectively. In contrast, no AFs G1 and G2 were detected. This study showed that the fungal infection of Indonesian dried chili occurs both in the field and during storage; thus, it is suggested to implement good agricultural and handling processes.

Pretreatment technologies for anaerobic digestion of lignocelluloses and toxic feedstocks.

Several feedstocks for anaerobic digestion (AD) have challenges that hamper the success of AD with their low accessible surface area, biomass recalcitrance, and the presence of natural inhibitors. This paper presents different types of pretreatment to address those individual challenges and how they contribute to facilitate AD. Organosolv and ionic liquid pretreatments are effective to remove lignin without a significant defect on lignin structures. To deal with accessible surface area and crystallinity, comminution, steam explosion, pretreatment using N-methyl-morpholine-N-oxide methods are suggested. Moreover, solid extraction, simple aeration, and biological treatments are capable in removing natural inhibitors. Up to date, methods like comminution, thermal process, and grinding are more preferable to be scaled-up.

Effect of Effluent Recirculation on Biogas Production Using Two-stage Anaerobic Digestion of Citrus Waste.

Citrus waste is a promising potential feedstock for anaerobic digestion, yet the presence of inhibitors such as d-limonene is known to limit the process. Effluent recirculation has been proven to increase methane yield in a semi-continuous process for recalcitrant material, but it has never been applied to toxic materials. This study was aimed to investigate the effect of recirculation on biogas production from citrus waste as toxic feedstock in two-stage anaerobic digestion. The first digestion was carried out in a stirred tank reactor (STR). The effluent from the first-stage was filtered using a rotary drum filter to separate the solid and the liquid phase. The solid phase, rich in hydrophobic D-limonene, was discarded, and the liquid phase containing less D-limonene was fed into the second digester in an up-flow anaerobic sludge bed (UASB) reactor. A high organic loading rate (OLR 5 g VS/(L·day)) of citrus waste was fed into the first-stage reactor every day. The effluent of the first-stage was then fed into the second-stage reactor. This experiment was run for 120 days. A reactor configuration without recirculation was used as control. The result shows that the reactor with effluent recirculation produced a higher methane yield (160⁻203 NmL/g·VS) compared to that without recirculation (66⁻113 NmL/g·VS). More stable performance was also observed in the reactor with recirculation as shown by the pH of 5⁻6, while without recirculation the pH dropped to the range of 3.7⁻4.7. The VS reduction for the reactor with recirculation was 33⁻35% higher than that of the control without recirculation. Recirculation might affect the hydrolysis-acidogenesis process by regulating pH in the first-stage and removing most of the D-limonene content from the substrate through filtration.

Semi-Continuous Reverse Membrane Bioreactor in Two-Stage Anaerobic Digestion of Citrus Waste.

The presence of an antimicrobial compound called D-Limonene in citrus waste inhibits methane production from such waste in anaerobic digestion. In this work, a two-stage anaerobic digestion method is developed using reverse membrane bioreactors (rMBRs) containing cells encased in hydrophilic membranes. The purpose of encasement is to retain a high cell concentration inside the bioreactor. The effectiveness of rMBRs in reducing cell washout is evaluated. Three different system configurations, comprising rMBRs, freely suspended cells (FCs), and a combination of both (abbreviated to rMBR⁻FCs), are incubated at three different organic loading rates (OLRs) each, namely 0.6, 1.2, and 3.6 g COD/(L cycle). Incubation lasts for eight feeding cycles at 55 °C. Methane yield and biogas composition results show that rMBRs perform better than rMBR⁻FCs and FCs at all three OLRs. Volatile fatty acid profiles and H2 production show that the reactors are working properly and no upset occurs. Additionally, a short digestion time of 4 days can be achieved using the rMBR configuration in this study.

Improvement of biogas production from orange peel waste by leaching of limonene.

Limonene is present in orange peel wastes and is known as an antimicrobial agent, which impedes biogas production when digesting the peels. In this work, pretreatment of the peels to remove limonene under mild condition was proposed by leaching of limonene using hexane as solvent. The pretreatments were carried out with homogenized or chopped orange peel at 20-40°C with orange peel waste and hexane ratio (w/v) ranging from 1 : 2 to 1 : 12 for 10 to 300 min. The pretreated peels were then digested in batch reactors for 33 days. The highest biogas production was achieved by treating chopped orange peel waste and hexane ratio of 12 : 1 at 20°C for 10 min corresponding to more than threefold increase of biogas production from 0.061 to 0.217 m(3) methane/kg VS. The solvent recovery was 90% using vacuum filtration and needs further separation using evaporation. The hexane residue in the peel had a negative impact on biogas production as shown by 28.6% reduction of methane and lower methane production of pretreated orange peel waste in semicontinuous digestion system compared to that of untreated peel.

Effects of lactone, ketone, and phenolic compounds on methane production and metabolic intermediates during anaerobic digestion.

Fruit waste is a potential feedstock for biogas production. However, the presence of fruit flavors that have antimicrobial activity is a challenge for biogas production. Lactones, ketones, and phenolic compounds are among the several groups of fruit flavors that are present in many fruits. This work aimed to investigate the effects of two lactones, i.e., γ-hexalactone and γ-decalactone; two ketones, i.e., furaneol and mesifurane; and two phenolic compounds, i.e., quercetin and epicatechin on anaerobic digestion with a focus on methane production, biogas composition, and metabolic intermediates. Anaerobic digestion was performed in a batch glass digester incubated at 55 °C for 30 days. The flavor compounds were added at concentrations of 0.05, 0.5, and 5 g/L. The results show that the addition of γ-decalactone, quercetin, and epicathechin in the range of 0.5-5 g/L reduced the methane production by 50 % (MIC50). Methane content was reduced by 90 % with the addition of 5 g/L of γ-decalactone, quercetin, and epicathechin. Accumulation of acetic acid, together with an increase in carbon dioxide production, was observed. On the contrary, γ-hexalactone, furaneol, and mesifurane increased the methane production by 83-132 % at a concentration of 5 g/L.

Performance of semi-continuous membrane bioreactor in biogas production from toxic feedstock containing D-Limonene.

A novel membrane bioreactor configuration containing both free and encased cells in a single reactor was proposed in this work. The reactor consisted of 120g/L of free cells and 120g/L of encased cells in a polyvinylidene fluoride membrane. Microcrystalline cellulose (Avicel) and d-Limonene were used as the models of substrate and inhibitor for biogas production, respectively. Different concentrations of d-Limonene i.e., 1, 5, and 10g/L were tested, and an experiment without the addition of d-Limonene was prepared as control. The digestion was performed in a semi-continuous thermophilic reactor for 75 days. The result showed that daily methane production in the reactor with the addition of 1g/L d-Limonene was similar to that of control. A lag phase was observed in the presence of 5g/L d-Limonene; however, after 10 days, the methane production increased and reached a similar production to that of the control after 15 days.

Biogas production from citrus waste by membrane bioreactor.

Rapid acidification and inhibition by d-limonene are major challenges of biogas production from citrus waste. As limonene is a hydrophobic chemical, this challenge was encountered using hydrophilic polyvinylidine difluoride (PVDF) membranes in a biogas reactor. The more sensitive methane-producing archaea were encapsulated in the membranes, while freely suspended digesting bacteria were present in the culture as well. In this membrane bioreactor (MBR), the free digesting bacteria digested the citrus wastes and produced soluble compounds, which could pass through the membrane and converted to biogas by the encapsulated cell. As a control experiment, similar digestions were carried out in bioreactors containing the identical amount of just free cells. The experiments were carried out in thermophilic conditions at 55 °C, and hydraulic retention time of 30 days. The organic loading rate (OLR) was started with 0.3 kg VS/m3/day and gradually increased to 3 kg VS/m3/day. The results show that at the highest OLR, MBR was successful to produce methane at 0.33 Nm3/kg VS, while the traditional free cell reactor reduced its methane production to 0.05 Nm3/kg VS. Approximately 73% of the theoretical methane yield was achieved using the membrane bioreactor.