Collection of brewer's spent grain and brewer's spent yeast

The brewer's spent grain and brewer's spent yeast were obtained from International Breweries Plc., Ilesha in Osun state, Nigeria. The town is located at longitude 7.6395°N and latitude 4.7588°E.

Collection of fruits from various locations in Osun State

Various types of fruits were collected from different locations in Osun State. This State was grouped into four (4) zones, A: Odeomu/Gbongan axis, B: Ife and its environment, C: Osogbo, and D: Ilesha and its environment. A total of 161 different fruits were collected as pineapple (Ananas comosus), orange (Citrus sinensis), African star apple (Chrysophyllum albidum), tomato (Solanum lycopersicum), banana (Musa acuminata), lime (Citrus aurantiifolia), pawpaw (Carica papaya), sour-sop (Annona muricata), watermelon (Citrullus lanatus), apple (Malus domestica), plantain (Musa paradisiaca) and almond (Prunus dulcis).

Source of S. carlsbergensis

S. carlsbergensis was isolated from brewer's spent yeast using serial dilution method. A loop full from 10^-6 was cultured on Potato Dextrose Agar (PDA) and incubated at 25 °C for 48 h. The yeast was subcultured into another freshly prepared PDA and incubated at 25 °C for 48 h to get pure isolate of S. carlsbergensis. The pure isolate was transferred to yeast broth and incubated for 48 h.

Production of bioethanol from BSG

The method, according to Alam et al. (2009), was adopted for the production of bioethanol with a slight modification. The fermenter was sterilized using 3% v/v of hypochlorite. The sterile fermenter was loaded with 50 g L^-1 of BSG at 29±2 °C and an agitation speed of 180 rpm. The pH was adjusted to 6.0 before the addition of 500 mL of yeast culture; the fermentation lasted 7 days under submerged and optimized conditions. The aeration and pH were kept stable during fermentation. The fermented product was concentrated using a rotatory evaporator at 66±1 °C, and ethanol was qualitatively determined by the dichromate method.

FTIR spectroscopic of bioethanol from BSG

Structural analysis of the functional group in bioethanol was determined using FT-IR spectroscopy (8400S, Shimadzu Scientific Instruments Inc.). BriefIy, bioethanol (1.0 µL) was placed on a fused KBr disc. This was placed on the cell holder, clamped loosely and fixed on the infrared (IR) beam. The running was done at 400 to 4000 per cm wavenumber.

Isolation and identification of microorganisms from fruits

The surface of fruit was sterilized with 1% v/v hypochlorite and rinsed with sterile distilled water to remove normal fIora of fruit and other possible microbial contaminants. Fruit samples were observed for 4-5 days to check any sign of spoilage. The rotten part of fruit was aseptically cut and transferred into sterilized peptone water. The sample was shaken vigorously and then allowed to stand for 30 min (Ajayi-Moses et al., 2019). Serial dilution was carried out up to10^-4 and 10^-5 dilution factor. An aliquot of 0.1 mL was aseptically transferred into Petri dish, and molten nutrient agar or PDA was then introduced. The plate solidified at room temperature (29±1 °C), then plates were incubated at 37 °C for 24 h and 2-3 days at 25 °C for bacteria and fungi, respectively. Discrete colonies were counted and recorded as colony-forming unit per gram (CFU g^-1) for bacteria and spore-forming unit per gram (SFU g^-1) for fungi. A pure colony was obtained by subcultured, and isolates were tentatively grouped according to their morphological, cultural, and staining characteristics. Biochemical tests such as the catalase test, production of hydrogen sulfide (H₂S), indole, urease, methyl red, oxidase, coagulase, motility, methyl red, Voges-Proskauer, starch hydrolysis and sugars fermentation were carried out using the methods described by Olutiola et al. (2000). The results of the biochemical test were compared to Bergey's Manual of Systematic Bacteriology (Krieg et al., 2010). Fungi isolates were identified using cultural and microscopic observations, according to Barnett et al. (2000) and Samson et al. (2010).

In vitro antimicrobial activity of bioethanol and other preservatives against microorganisms

The antimicrobial activity of the bioethanol against spoilage microorganisms isolated from fruits was performed using agar well diffusion (CLSI, 2014). Suspension of test microorganisms was adjusted by the spectrophotometer to 0.5 McFarland standard. Sterile cotton swabs were dipped in the microbial suspension and spread on the surface of the agar plate. A sterilized cork borer was used for cutting wells in each plate. Bioethanol (50 μL) was introduced and sorbic acid (5.0 mg mL^-1), ampiclox (5.0 mg mL^-1) and terbinafine (5.0 mg mL^-1) were implemented as positive controls against bacteria and fungi, while sterile distilled water was used as the negative control. All the plates were labeled appropriately and incubated at 37 °C during 24 h for bacteria and at 25 °C for 48 h for fungi. The diameter of zones of inhibition around wells was measured in millimeter (mm).

Preservation of fruits using bioethanol from BSG

Most prevalent as well as colonizing microorganisms (1.0×105 CFU mL-1 or SFU mL-1) were re-introduced to apparently healthy (absence of diseases, no wound symptoms or lesion) selected fruits (orange, watermelon, pineapple, and tomato). Each fruit was discretely remained in a sterile laminar hood without touching each other. After 24 h, bioethanol (20 mL) was applied on the surface fruits of the group A. The bioethanol was adjusted to 40% v/v since Kalathenos and Russell (2003) revealed that <30% v/v were rarely biocidal. Ethanol ≥70% v/v could cause damage to the fruit integument. Group B was treated with sorbic acid (5% w/v), group C was un-inoculated fruits with microorganism and group D was fruits inoculated with microorganisms but untreated with either bioethanol or sorbic acid. The fruit samples were observed for 7 days at room temperature (29 °C).

Statistical analysis

Data were presented as mean±standard deviation from three repetitions. Data obtained in this study were subjected to One-way Analysis of Variance (ANOVA) using Statistical Package for Social Sciences (SPSS) version 20 (USA). For bacteria and fungi count, the mean values were compared by Duncan's new multiple range test (MRT). Differences were considered significant at P<0.05.

Yield and physicochemical parameters of bioethanol from BSG with S. cerevisiae

Table 1 shows the physicochemical parameters of bioethanol produced from BSG. The yield obtained was 22%, with a specific gravity of 0.8. These results contrast with the findings of Irfan et al. (2014). They reported bioethanol from sugarcane bagasse, rice straw and wheat straw by S. cerevisiae with a value of 77 g L-1, 62 g L-1, and 44 g L-1, respectively. Ingale et al. (2014) obtained ethanol of 17.1 g L-1 with a higher yield of 84% from banana pseudostem fermented by S. cerevisiae NCIM 3570. The higher proportion of ethanol in their study could be associated with two fungal strains of Aspergillus spp., which facilitated the maximal release of sugars to produce more ethanol. The combination of Aspergillus oryzae and S. cerevisiae NCYC479 produced the highest concentrations of ethanol (37 g L-1) in 10 days from BSG using consolidated bioprocessing (Wilkinson et al., 2017). Moodley and Gueguim Kana (2019) optimized pretreatment techniques to produce 25% more bioethanol from sugarcane leaf waste using S. cerevisiae BY4743. S. cerevisiae is one of the best yeast widely employed for commercial production of bioethanol. S. cerevisiae has been attractive for efficient consolidated bioprocessing and several biotechnological purposes because of its novel amylolytic enzyme combination, relatively high tolerance to osmotic stress and anaerobic conditions. Therefore, it is suitable for large-scale fermentation of agro-wastes into bioethanol (Cripwell et al., 2019). In the study of Wu et al. (2020), replacement of ethanol fermentation-associated regulatory gene in S. cerevisiae was reported to enhance ethanol production by a 5.30% increase in yield and 12.5% decrease in fermentation time when compared to the original strain. Another method that could increase the yield of ethanol is mixed substrates. Bolade et al. (2019) claimed that multi-substrates biomass of agro wastes increases the yield of bioethanol.

Table 1: Physicochemical properties of bioethanol produced from BSG.

BSG is interesting biomass with hydrolyzable fermentable sugars that can be converted to ethanol with different microorganisms through co-fermentation strategies (Rojas-Chamorro et al., 2020). Likewise, cassava peels, potato peels and millet husks with different microbial inoculants such as S. cerevisiae, Rhizopus nigricans, Aspergillus niger, Spirogyra africana showed great potential for bioethanol production (Chibuzor et al., 2016). The large proportion of lignocellulosic materials such as corncob, cornstalk, cornhusk, sugarcane bagasse and sugarcane bark that are creating environmental pollution, can be easily degraded by microorganisms and thus, serve as a substrate for renewable resources (bioethanol).

Figure 1 shows the transmittance and peak representing functional groups in the bioethanol. Table 2 shows various functional groups found in bioethanol, hydroxyl (OH _stretch), methyl (CH_{3 stretch}), and alkane (CH_{2 stretch}). These functional groups in ethanol (alcohol) give it the biocide property, and it is responsible for the antisepsis, disinfection and can be used as a preservative agent (McDonnell and Russell, 1999). Hydroxyl group in ethanol (alcohol) acts as an antimicrobial agent against microbes except for alcohol-tolerant strains. It causes a partial breakdown of membrane function, inhibiting cell growth or protein synthesis, denaturation of proteins, and membrane damage, which lead to cell perturbations like ion leakage or loss of energy (Horinouchi et al., 2018).

Figure 1: FTIR spectrum of bioethanol produced from BSG

Table 2: The functional group identified in bioethanol produced from BSG.

Total microbial load and occurrence (%) of microorganisms isolated from selected fruits

The colonies counted from each fruit are shown in Table 3.

Table 3: Bacteria and fungi count from fruits obtained from different regions in Osun State.

The highest colonies of bacteria count (1.05×10⁵ CFU g^-1) were recorded for bananas from Odeomu (A) market (P<0.05). Pineapple from Ilesha had the highest fungal count (1.96×10⁶ SFU g^-1). Watermelon from Odeomu (A) had the least bacterial load (P<0.05) of 1.0×10⁴ CFU g^-1, and African star apple collected from Osogbo (C) had the least fungal count of 1.1×10⁵ SFU g^-1. Ajayi-Moses et al. (2019) reported the highest bacterial count of 5.84×10⁵ CFU g^-1 for tomatoes and African star apple with the highest fungal count of 3.04×10⁵ SFU g^-1. Evaluation of microorganisms associated with fruits revealed that various bacteria and fungi in high densities could spoil fruits. Spoilage microorganisms can also colonize, enter and penetrate plant tissues at fruit development, either through calyx, stem or various specialized water and gas exchange structures of leafy matter. However, successful establishment requires the spoilage microbe to overcome multiple natural protective barriers and other factors (Erkmen and Bozoglu, 2016). Lime and soursop did not show any sign of spoilage, and no microorganisms were isolated (Table 3). Fruits like lime and soursop with higher pKa do not get spoilt but rather get dehydrated, exiting fruit juice, a process attributed to spoilage organisms affecting fruits (Czajkowski et al., 2011).

Table 4 shows the percentage of the occurrence of bacteria. The highest percentage of occurrence (25.6%) was obtained for Corynebacterium sp. followed by Lactobacillus sp. with 17.9%. The lowest bacteria percentage occurrence (5.1%) was obtained for species of Micrococcus and Staphylococus. Erwinia spp. were found to be associated with spoilage of pineapple, African star apple, tomato, watermelon and almond. The bacterium Erwinia carotovora subsp. carotovora is a highly effective spoilage microorganism that produces an increasing amount of pectolytic enzymes to degrade fruit tissues. It causes soft rot on fruits like oranges, tomatoes, banana, pineapple, and watermelon (Barth et al., 2009; Sharma et al., 2013). Besides, E. carotovora, several Pseudomonas spp., Corynebacterium, Xanthomonas campestris and lactic acid bacteria are important spoilage bacteria of fruits (Tournas, 2005; Erkmen and Bozoglu, 2016). Some spoilage microbes are capable of colonizing, creating lesions and damaged healthy plant tissues. The type of microbial spoilage in fruits is based on the pH, nutrient availability, water activity (a_w), temperature, relative humidity, oxidation-reduction potential and content of biological structure of fruits (Erkmen and Bozoglu, 2016).

Table 4: Percentage of occurrence of bacteria isolated from different locations

Fungi with the highest percentage of occurrence was Aspergillus niger (13.9%), followed by Colletotrichum sp. and Penicillium digitatium with the same value of 11.1%. Pichia sp. has the lowest percentage of occurrence of 4.2% (Table 5). The highest occurrence of A. niger from examined fruits in Osun State is in concordances with findings of Mailafia et al. (2017). Researchers revealed that Aspergillus spp. had the highest occurrence in fruits like pineapple, watermelon, oranges, pawpaw, and tomatoes with a frequency of 38%, followed by Fusarium avenaceum with occurrence of 31% in pineapple, watermelon, oranges, pawpaw and tomatoes. In comparison, P. digitatum and R. stolonifera have the least frequency at the same value of 4% for tomato and orange. Other fungal species identified as agents of spoilage were Saccharomyces spp. (10%), F. solani (8%), and A. flavus (5%). Some of the fungi isolated from fruits were species of Penicillum, Aspergillus, Rhizopus, Fusarium and Mucor iriformis. Tafinta et al. (2013) and Ajayi-Moses et al. (2019) isolated similar microorganisms with varying prevalence from banana, pawpaw, orange, tomato, apple, pineapple, watermelon, cucumber, and African star apple. Some of these fungal isolates are known to be pathogenic due to the toxic secondary metabolites produced. Penicillium expansum and Botrytis cinerea are pathogenic spoilage microorganisms, which cause blue-rot, grey mold in African star apple, cherry, apple, tomato, pears and kiwi fruit called sour sop (Miedes and Lorences, 2004).

Table 5: Percentage of occurrence of fungi isolated from different locations

Likewise, molds of genera Rhizopus, Alternaria and Botrytis produce acidic compounds that cause fruit and vegetable rot with distorted color, texture and or taste (Tournas, 2005). Fernández-Cruz et al. (2010), Lewis and Goodrich-Schneider (2012) revealed different species of fungi that produce mycotoxins (aflatoxins, ochratoxin, patulin, fumonisin, alternariol, alternariol methyl ether, and altenuene) in fruits. The problem associated with mycotoxins in fruits includes economic loss, poor organoleptic properties, toxicities (acute to chronic), and a spectrum of effect (mild to severe), including carcinogenicity and death. Spoilage microorganisms exploit fruit components using their extracellular lytic enzymes, pectinases and hemicellulases to degrade fruit polymers to release intracellular constituents as nutrients for growth (Kalia and Gupta, 2006). Microorganisms from fruits secreted a wide variety of enzymes. Isolation and identification of novel strains of microorganism from fruits can serve as natural origin, a safer and cheaper alternative source of microbial enzymes as promising candidates for biotechnological uses and medical processes (Sharma et al., 2013; Garg et al., 2016).

Antimicrobial and preservative properties of bioethanol from BSG

To minimize wastage recorded on fruits and vegetables and to reduce economic losses associated with microbial spoilage, a reliable and supportive measure needs to be sourced. Bioethanol from BSG displayed zones of inhibition against bacteria with values ranging from 7.0 to 8.8 mm, sorbic acid was within 6.0 to 13.0 mm, and ampiclox was from 8.0 to 18.5 mm (Table 6). Zones of inhibition by bioethanol against fungi ranged from 7.5 to 11.5 mm, while sorbic acid was 7.5 to 13.0 mm and 10.0 to 17.0 mm for terbinafine (Table 6). Ethanol is used as a disinfectant and acts against microorganisms in two different ways: growth inhibition (bacteriostasis, fungistasis) or lethal action (bactericidal, fungicidal or virucidal effects) (Maris, 1995). Hence, bioethanol can be used to prevent the growth of spoilage microorganisms on fruits. Ethanol interacts with cell surface followed by penetration into cells and acts on the target site(s) of microorganisms (Maris, 1995). Inactivation of fungal spores, suppression of fungal growth or their germination on fruits by ethanol reflects the inhibitory potential of bioethanol in controlling fruit decaying by fungi and extending the shelf-life of food products (Dao and Dantigny, 2011).

Table 6: Zones of inhibition (mm) displayed by bioethanol and selective preservatives against #microorganisms isolated from fruits.

Table 7 shows different fruits preserved with bioethanol, sorbic acid, un-inoculated fruit with the microorganism, spoilt fruit and open spoilt fruit after 7 days. Dao and Dantigny (2011), found that oranges were protected from fungal infection for 30 days when exposed to 20-100% ethanol. The use of bioethanol as a preservative is important to prevent a higher loss of fruits since it has no side effects on humans. It will proffer the solution to longstanding spoilage of vegetables and fruits, which had been associated with different microorganisms. Ethanol exerts its most effective bactericidal action at ≥40 to 95% v/v against vegetative cells like chemical disinfectants (Kalathenos and Russell, 2003). Findings of Katsinis et al. (2008) established effective preservation with the synergistic effect of conventional chemical preservative (potassium sorbate) and ethanol, which improved the shelf life of bread by suppressing microbial growth (43.5% and 38.5% mold-free shelf life). Bioethanol displayed a higher zone of inhibition (11.5 mm) against Saccharomyces sp. than sorbic acid (8.6 mm). Sorbic acids had less or no inhibitory activity against S. cerevisiae, Saccharomycodes ludwigii, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Schizosaccharomyces pombe Brettanomyces spp., Pichia membranifaciens, Dekkerae spp, and Issatchenkia orientalis at 800 mg kg^-1, which is much higher than the permissible limit in foods, but ethanol (>20% v/v) exerts a strong lethal effect on these molds (Loureiro and Malfeito-Ferreira, 2003).

Table 7: Evolution of fruits during 7-day treatment with bioethanol and sorbic acid

The mechanisms of resistance by yeasts to weak organic acids are by inducing the expression of H⁺ ATPases to regulate their cytosolic pH, using their plasma membrane components to modulate the influx of lipophilic weak organic acids (Ullah et al., 2012). ATP binding cassette (ABC) transporter (Pdr12) prevents anion accumulation or degrade sorbic acid to 1,3-pentadiene (Casas et al., 2004). Findings of Linares-Morales et al. (2018) revealed that chemical preservatives like organic acids in fruits and vegetables cause disruption of membrane permeability, reduce cell's internal pH, affects metabolic enzymes as well as protein synthesis. However, a microbial product like bioethanol can be encouraged in use as preservative agent since it strongly suppresses microbial activity on fruits. Microbial by-products are bio-protective or natural preservatives, and thus, exhibit antifungal activities. There is a growing interest in alternatives to preservation other than chemical or synthetic agents with different side effects (Leyva Salas et al., 2017). The microbial growth inhibition or killing action of bioethanol on microorganisms is an indication that; bioethanol produced by S. cerevisiae from renewable biomass (lignocellulosic wastes) is needed for commercial purposes.