Abstract

The aim of this study was to add value to cashew apples from northern Côte d'Ivoire. Yeasts were isolated from cashew apples and screened to select potential starters. Following screening, the best strains underwent biochemical identification by MALDI TOF mass spectrometry to determine the species involved. The results enabled us to select three (3) strains with the best properties compared with the other strains tested. These three strains belong to the Saccharomyces cerevisiae species, according to their MALDI-TOF MS profiles. These highly interesting technological properties point to the potential use of these three strains as starters in biotechnological applications.

1. Introduction

The cashew tree (Anacardiumouest L.) has two fruit parts: the nut, which is the seed contained in a hard shell, and the cashew apple, a fleshy pseudo-fruit to which the nut is attached. Cashew nuts are traded internationally. Indeed, it is one of the world's most important agro-industrial products. Global cashew nut production is estimated at over 3.7 million tonnes, with Côte d'Ivoire, the world leader in the production ¹. In Côte d'Ivoire, the surface area of cashew orchards is estimated at over 1,400,000 ha belonging to more than 420,000 producers, with production rising from 702,000 tonnes of raw cashew nuts in 2015 to 1,024,000 tonnes in 2022 ². The cashew apple is 9 to 10 times the weight of the nut, corresponding to over 6 million tons of cashew apples. Almost all of this production is lost at the point of harvest because it is not used industrially in Côte d'Ivoire due to its astringency and certain taboos ³. Abandoning apples in the fields is a source of environmental pollution. Cashew apples are very rich in vitamin C and fermentable sugars ^{4, 5}. This makes it a suitable substrate for yeast. Thus, the search for new yeast strains from natural sources such as cashew apple could be of interest to the food industry. These new strains could offer interesting properties, such as improved tolerance to environmental stresses or enhanced production of compounds of interest to the food industry ⁶. Other studies have shown that yeasts can be used to produce fermented beverages with unique flavor profiles ^{7, 8}. The aim of this study is to characterize cashew apple yeasts with a view to using them for biotechnological applications.

2. Materials and Methods

The biological material was yellow cashew apples harvested at maturity (Figure 1) from a farmer's field at Foro in the sub-prefecture of Tioroniaradougou (Korhogo Department, Côte d'Ivoire). The apples were taken to the laboratory of the Peleforo GON COULIBALY University in KORHOGO for microbiological analysis.

Twenty-five (25) grams of cashew apples are diluted in 225 ml of peptone salt solution (0.1% (w/v) bactopeptone and 0.85% (w/v) NaCl). The solution thus prepared constitutes the stock solution, which undergoes successive decimal dilutions (10^-1 to 10^-4) with tryptone salt solution. A volume of 100 μ L of each dilution has been streaked onto MYGP agar (3 g/L yeast extract, 3 g/L malt extract, 5 g/L bactopeptone and 10 g/L glucose) containing 100 mg/L chloramphenicol (Sigma). After inoculation, Petri dishes have incubated and yeast strains have morphologically been identified after 3 days of incubation at 30°C, then yeast cells have been observed fresh under a precision optical microscope (Zeiss Microlmaging GmbH 37081, Germany) at objective ˟100. Presumptive yeast isolates were stored in cryotubes containing MYGP broth supplemented with 20% glycerol at -20°C for subsequent testing ⁹.

Depending on the test to perform, strains are grown overnight at 25 ◦C either on MYGP agar or in MYGP broth and then the cultures are used to inoculate either MYGP broth or specific media. For the latter, each strain is harvested by centrifugation (5000 rpm for 10 min), washed once in NaCl 0.9% (w/v) solution, re-suspended to Optical Density (OD) 600 of 1.0 in the same solution ¹⁰. Subsequently, each strain has been spotted (5 µL) in duplicate onto specific media.

The fermentative capacity of yeast strains isolated from cashew apples has been studied according to the method of ¹¹ with a slight modification. From pre-culture of 24 hours, pure yeasts culture has been suspended in saline tryptone to get an optical density of 0.7 at 600 nm and 100 µL of this suspension have been used to inoculate 10 mL of YPG medium containing a Durham tube into essay tube. Then, the culture has been incubated at 30°C for 6 days, without agitation. Fermentative capacity has also been determined by measuring the gas production in Durham test. Yeasts usually oxidize sugars into ethanol with production of gas ¹². This volume of gas and ethanol produced is related to the fermentative strength of strain ¹³.

The method described by ¹⁴ was used to demonstrate the catalase activity of yeast isolates. Yeast biomass, sampled with a 1 μl sterile loop, was added to a drop of 3% (v/v) H ₂ O _2. The development of bubbles has indicated positive activity.

A loopful (1 µL) of biomass of each strain has been streaked onto Chalk agar (yeast extract 3 g/L, glucose 10 g/L, calcium carbonate 3 g/L, agar 15 g/L) plates and has incubated for 7 d at 25°C [ 15]. The presence and extent of a clear halo around the yeast biomass has indicated the rate of acetic acid production.

The method described by ¹⁶ was used to identify the protease activity of yeast isolates. One hundred (100) ml of MYGP medium (3 g/L malt extract, 3 g/L yeast extract, 5 g/L bacteriological peptone, 10 g/L glucose, 5 g/L NaCl and 20 g/L agar) were prepared and sterilized for 15 min in an autoclave at 121°C. Separately, 100 ml of skimmed milk solution (10% w/v) were prepared and autoclaved at 121°C. The two media were mixed and poured into Petri dishes. Isolates were then spot-inoculated and incubated at 30°C in the oven for 48 hours. The presence of a clear zone around the colony indicates the presence of protease activity.

To assess hydrogen sulphide production, 10 µL of each strain from the previously prepared YPD liquid were inoculated on the BIGGY medium and kept for 2–3 days at 24 C, respectively. Visual scale was used as a function of the increasing level of H ₂ S produced ^{17, 18}.

Yeast strains have been grown in standard liquid medium containing 0.05% yeast extract; 0.3% casein peptone; 1% glucose at pH 5.6. To assess the influence of temperature on the growth of yeast isolates, 10 ml of standard liquid medium contained in a test tube has been inoculated with 100 µL of yeast pre-culture, OD600 = 0.7. The cultures have then been incubated for 72 h at temperatures ranging from 30 to 50°C. The influence of pH variations on the growth of yeast isolates has been analyzed in the same medium at different pH values (2.5; 4; 5 and 7) and has been incubated at 30°C. Yeast isolate growth has been determined by measuring the turbidity of the culture medium at 600 nm using a spectrophotometer ¹⁰.

Ethanol assay was carried out at the end of fermentation. Fermented molasse was distilled to ethanol, using the powerful Quickfit/FC3/13 column distiller, 85 cm long and 4.45 cm in diameter (Fischer Scientific, Sweden) A temperature of 79°C was maintained at the top of the column until all the alcohol in the fermented juice was evaporated and condensed. Ethanol content was determined using an alcoholmeter (Biobase, china). Three independent experiments were carried out.

The yeast cultures isolated from the cashew apple extract were prepared as per the method developed by ¹⁹. Direct colony transfer - formic acid method was adopted for preparing the isolated yeast cultures in feeding the MALDI-TOF MS. Young 24 hours grown east isolates (CAP 1 to CAP 9) were smeared separately on a polished steel MSP 96 target plate using a tooth pick on which 1 µL of 70% formic acid was added, and thin smear was prepared allowed for air drying. Followed by it, saturated-cyano-4-hydroxycinnamic acid (HCCA) matrix (1 µL) prepared using 50% acetonitrile and 2.5% trifluoroacetic acids solution was overlaid onto the air-dried yeast isolates on the target plate at room temperature. The target plate was placed into the plating chamber of the MALDI-TOF MS and shuttered for performing the identification of the yeasts.

All measurements have been performed in triplicate. Statistical analyzes of the data have been performed through Statistica version 7.1 software. Means have been compared through Tuckey's HSD test with a significance level of only 5% (p < 0.050).

3. Results and Discussion

forty-one (41) yeast isolates have been isolated from cashew apples. Table 1 shows the morphology, color, and texture of the colonies of the different strains grown on MYGP agar.

Among the 41 yeast isolates tested for their fermentative capacity, only nine (9) isolates or 21.95% showed a high fermentative capacity with a CO2 volume equal to 7.5 cm3. While ten (10) isolates or 24.39% produced CO2 moderately with values ranging from 1 to 4 cm3 and twenty-two (22) isolates or 53.66% produced CO2 weakly with values from 0 to 1 cm3 (Table 2). Highly fermentable isolates (9) were selected to study their biochemical properties.

All the yeasts isolates involving cashew apples fermentation tested have the catalase and H ₂ S production capacity. But the level of production is different from one yeast isolate to another. About protease activity, seven (7) have produced with high activity for three (3) isolates (YAN 2 YAN 36, YAN 41). While only tree (3) yeasts isolates (YAN 9, YAN 20, YAN 39) are able to produce acetic acid (Table 3).

The results show a significant difference in isolate growth at different pH levels (2.5, 4 and 7). However, at pH 5, isolate growth showed no significant difference (Table 4). All isolates tested showed good growth at pH 4. At this pH, isolate YAN36 had the best growth (0.966) and isolate YAN had the lowest growth (0.691)

Yeast isolates showed varying growth rates at different temperatures. Statistical analyzes reveal a significant difference in the growth of isolates. The result of the growth of the 9 best yeasts isolates with a high fermentation power at different temperature give two. The first group consists of yeast isolates (YAN40, YAN20 and YAN39) with shows the best growth at 30° C. The second group consists of yeast isolates (YAN2, YAN41, YAN36, YAN35, YAN9 and YAN11) which shows good growth at 35°C. Three isolates, YAN2, YAN41 and YAN36, grew at 45°C.

Isolates YAN 2, YAN 36, and YAN 41 showed the best characteristics (high CO ₂ production, no acetic acid production, high catalytic activity, high protease activity, low H ₂ S production, and good resistance to temperature and pH variation) for fermentation. These isolates have been selected for ethanol production and identified. The three selected isolates were all identified as Saccharomyces cerevisiae strains on the basis of their MALDI-TOF MS profiles. The ethanol production of selected isolates YAN2, YAN36, YAN41 was 6.39±0.12%, 6.44±0.09% and 6.42±0.09% respectively (Figure 2). Statistical analysis showed that there was no significant difference between the ethanol production of the three yeast strains.

4. Discussion

The study of technological properties is essential in the search for strains with exceptional properties ²⁰. A study of the fermentative power of yeasts isolated from cashew apples revealed that 21.95% of isolates had a high fermentative power with a volume of 7.5 cm3 of CO2 produced. These results differ from those of ¹⁰ who isolated yeasts with high fermentation capacity from Cola Cordifolia pulp, but with CO2 volumes of between 4 and 6 cm3.

As far as the study of pH is concerned, its variation had a significant influence on the growth of yeast isolates with high fermentation capacity. Indeed, the optimal pH for growth of the yeast strains isolated was pH 4. These results differ from those of ²¹ whose strains perform best at pH 3. Similarly, temperature variation influenced the growth of yeast isolates. Temperatures above 40°C had a negative influence on the growth of yeast isolates with high fermentation capacity. Indeed, temperature is the main parameter influencing the activity and physiology of microorganisms ²². It affects growth rate, CO2 production rate, ethanol production and cell viability ²³.

The biochemical properties important for optimal fermentation were evaluated. Three isolates (YAN2, YAN36, YAN41) showed interesting biochemical properties, namely high catalase activity, protease production and inability to produce acetic acid. Indeed, high catalase activity is a property that enables strains to cope with oxidative stress during fermentation ²⁴. The production of proteases by yeast strains is essential and plays a significant role in protein degradation. During fermentation, proteases released by yeast play a role in the degradation of proteins present in the fermentation medium ²⁵. Protease is also one of the yeast enzymes of interest, like esterases, glycosidases and cellulases, capable of hydrolyzing structural components ²⁶. The results are similar to those of ²¹ who showed a protease property of a Saccharomyces cerevisiae strain involved in cocoa fermentation. Moreover, acetic acid production by yeasts during alcoholic fermentation is not desirable ²⁷. The inability of selected isolates to produce acetic acid is therefore of great interest. Similarly, the production of hydrogen sulfite by strains is unfavorable to fermentation. Thus, yeast strains producing little or no H2S are particularly important, as there is no sulfite formation during fermentation. The difference in H2S production by yeast isolates obtained in this study has been confirmed by several authors ^{28, 29}. The three selected isolates with the best characteristics are from the Saccharomyces cerevisiae species. However, ³⁰ Gayathrya et al (2023) identified by MALDI-TOF MS, four yeast strains namely Brettannomyces bruxellensis, Candida krusei, Candida tropicalis, Pichia norvegensis that colonize cashew apple in India. This difference could be explained by the geographical location, variety and degree of ripeness of the apples.

With regard to ethanol production, the ethanol production of the Saccharomyces cerevisiae strains was 6.39 ± 0.12% for strain YAN2, 6.44 ± 0.09% for strain YAN36 and 6.42 ± 0.09% for strain YAN41. The ethanol yields of the three strains were statistically identical and close to those of ²⁰ whose yields ranged from 5.39 to 6.81% for Pichia manshurica, Hanseniaspora uvarum and Candida parapsilosis species.

5. Conclusion

In all, 41 yeast strains were isolated from cashew apples, nine (9) of which were highly fermentative. High-fermenting isolates showed good growth at different pH levels and temperatures between 30 and 40°C. The biochemical properties of high-fermenting isolates vary depending on the isolate. Three isolates showed the best technological properties and were identified as Saccharomyces cerevisiae strains. These strains, with their interesting technological properties, hold great promise for biotechnological applications.

References