1 Introduction

Yeasts, being eukaryotic organisms, possess a remarkable capacity to synthesize an extensive repertoire of enzymes that are widely regarded as environmentally benign enzymes, and thus find diverse applications across various domains. The capacity of yeasts to generate lipase, amylase, and protease enzymes embodies essential attributes in the domain of food ecosystems; these enzymes assume a pivotal function in yeast colonization and can influence their interactions with other microorganisms [1, 2]. The microorganisms that thrive on the surface of cheese exert an influence on the procedure of maturation through the activity of lipase and protease enzymes. Yeasts contribute to the maturation process, both directly and indirectly [3,4,5].

Pectinases, being enzymes, are accountable for the degradation of pectin substances. These enzymes are well-known for their environmentally friendly and sustainable characteristics, and they have a wide range of potential applications in various industrial processes, such as the manufacturing of tea and coffee, the extraction of oil, and the filtration of juice [6]. In the course of coffee fermentation, the enzymes poly galacturonase (PG), pectin lyase (PL), and pectin methylesterase (PME) display distinct features [7]. These primary pectinase enzymes possess the capability to fully dismantle pectin, resulting in the production of galacturonic acid and its corresponding oligomers [8, 9].

β-galactosidase, also referred to as lactase, functions as an enzymatic catalyst that facilitates the process of hydrolytic cleavage of lactose into its constituent mono saccharides, namely glucose and galactose, by means of the breakdown of the β-1,4 linkage which connects galactose and glucose [10]. Lactase assumes a pivotal role in numerous domains, encompassing research, bioremediation, diagnosis, and the food industry [11].

Three fundamental classifications have been identified for issues related to lactose, encompassing those related to the technology of food, specifically the formation of lactose crystals; concerns regarding health, such as lactose intolerance; and considerations regarding the environment, as indicated by the significantly increased demand for biochemical and chemical oxygen in whey, the main source of lactose.

β-galactosidase, due to its remarkable activity in transgalactosylation, is commonly utilized in the production of galactooligosaccharides with prebiotic properties [12]. Microorganisms that possess the ability to produce urease can break down urea into ammonia and carbon dioxide, and can be isolated from various ecosystems. This enzyme has been extensively employed in a wide range of fields, including biotechnology, medicine, agriculture, and geotechnical engineering [13].

The 18S rRNA gene is frequently utilized as a marker to investigate the community composition of microorganisms belonging to the eukaryotic domain [14, 15]. The classification of fungi is often based on the determination of the sequence of the 18S rRNA gene using primers that are specific to fungi. With the proliferation of sequences in public databases, this approach has yielded novel insights into the diversity of fungal groups and has necessitated revisions in the taxonomy of fungi [16]. The utilization of phylogenetic analysis has proven to be a valuable approach employed in fungal investigations, which relies on the examination of the 18S rRNA gene sequences [17].

Consequently, the present work aims to isolate promising yeasts which have capacity to produce multi hydrolytic enzymes (pectinase, amylase, urease, lipase, β-galactosidase, and protease) which are known for their environmentally friendly and sustainable nature, and possess a vast range of potential applications in many purposes. The yeast isolates will be identified by using 18 s rDNA analysis after DNA yeast isolation and will be registered in the gene bank.

2 Materials and methods

2.1 Isolation and morphological characteristics of yeast isolates

Isolation was performed on a medium known as yeast extract peptone dextrose agar (YEPDA), whereby the yeast and soil samples were subjected to aerobic conditions and a temperature of 28 °C for duration of 72 h. The specimens utilized for this purpose exhibited variability in terms of their food source and soil composition. The evaluation of yeast colonies encompassed a comprehensive assessment of their color, surface characteristics, shape, and elevation. To gain insight into the morphology and arrangement of the yeast cells and budding, the technique of Gram staining was employed. Furthermore, a motility test was conducted to assess the yeast's capability to move, which was accomplished through the utilization of the hanging drop preparation test.

2.2 Screening of enzyme activity for yeast isolates

Yeast isolates were examined with regards to their enzymatic activity. Inoculation of a single location with a loop of yeast isolate was carried out into various media that contained suitable substrates for enzyme activity. To ascertain the activity of amylase, the isolates were applied to yeast extract and malt medium (YM) that was supplemented with glucose and starch [18]. The presence of pectinase enzymatic activity was observed by introducing isolates into a mineral medium that contained citrus pectin [19]. For the detection of protease activity, the yeast isolates were introduced into a basic medium that consisted of 1.0 g per liter (g/L) of skimmed milk as a protein source [20].

All isolates were cultured in (g/L): 5.0 peptone, 3.0 yeast extract, 20.0 agar, and 1.0 tributyrin, which were adjusted to pH 6.0, and incubated at 30 °C/48 h to determine their ability to produce the lipase enzyme [21].

For the determination of urease activity, a culture medium was prepared in accordance with the original formula proposed by Christensen [22]. The composition of the medium consisted of the following components, measured in grams per liter: peptone 1.0, NaCl 5.0, mono potassium phosphate 2.0, phenol red 0.012, glucose 1.0, and urea 20.0. In order to ensure sterility, the medium underwent autoclaving at a temperature of 121 °C for duration of 15 min, with the exception of the urea component. Prior to its addition to the medium, the urea was dissolved in 100 ml of distilled water and subsequently sterilized through filtration using a pore size of 0.22-lAm. Following the sterilization process, a loop of yeast isolates was inoculated into the medium and incubated at a temperature of 30 °C for a period of 48 h.

The presence of amylase and pectinase activities was assessed utilizing Lugol’s solution. The formation of a clear halo surrounding the colony was observed against a background of blue and brown, respectively [18, 19]. In the case of protease activity detection, the presence of a clear halo surrounding the colony was employed as an indicator [20].

The determination of lipase activity was conducted by observing the formation of a precipitation halo surrounding the colony. Urease activity was identified by the presence of a pink halo surrounding the colony. Yeast isolates that displayed clearing zones for amylase, protease, and pectinase enzymes were categorized as positive, as well as those that exhibited a precipitation zone in relation to the lipase enzyme.

To evaluate the β-galactosidase activity at a fundamental level, the isolates were introduced into test tubes that contained Durham tubes, 10 mL of YLP broth medium, and phenol red indicator at a pH of 6.0. These tubes were then incubated for a period of 3 days at a temperature of 25 °C on a rotary shaker with a speed of 120 rpm. The production of acid and gas was contingent upon the specific sugar consumed. Meticulous observation was carried out to monitor changes in pH, gas production, and the color of the medium.

The isolates that were selected from the primary screening in order to generate β-galactosidase underwent a secondary screening procedure. This subsequent screening process involved the quantitative estimation of the overall reducing sugars present in the culture. A total of eight yeast isolates that showed positive results were placed in 250 ml flasks containing 50 ml of YLP, which is a broth medium with a lactose concentration of 40 g per liter. These flasks were then positioned in a shaking incubator and incubated at a temperature of 30 °C and a speed of 180 rpm for durations of 72, 96, and 120 h, respectively. Following the incubation period, the cells were collected by means of centrifugation at a speed of 5000 rpm for 20 min at a temperature of 4 °C.

The supernatants were subsequently gathered and prepared for the assessment of the total reducing sugars via the employment of the phenol–sulfuric acid method. To carry out this method, 1 ml of the sample supernatant was introduced into a colorimetric tube. Subsequently, 1 ml of a 5% phenol solution was added, followed by the quick addition of 5 ml of concentrated sulfuric acid onto the surface of the mixture. The tube was then subjected to shaking via a vortex for duration of 1 min. After allowing the tubes to remain at room temperature for a period of 10 min, the absorbance of the resulting yellow-orange hue was measured at a wavelength of 480 nm. A blank was prepared by substituting the supernatant with distilled water, and a control was prepared using YLP broth medium without any isolate. In order to reduce errors, all sample solutions were prepared in triplicate.

2.3 Molecular characterizations of selected yeast isolates

2.3.1 Isolation of genomic and amplification of ITS region

The ABT DNA mini extraction kit, developed by Applied Biotechnology Co. Ltd in Egypt, was employed to extract nucleic acid from yeast. The amplification of the Internal Transcribed Spacer (ITS) region of the 18S rRNA genes was executed through the utilization of universal primers ITS1 and ITS4 (ITS1-primer: 5′-TCC GTA GGT GAA CCT GCG G -3′ and ITS4-primer: 5′-TCC TCC GCT TAT TGA TAT GC -3′) [23, 24]. Following the amplification process, the resultant PCR product underwent purification using the Wizard RSV gel and PCR clean-up system, provided by Promega (catalog number A928). Subsequently, sequencing of the approximately 600 bp long purified PCR product was carried out by lab technology services in Korea. This process was facilitated by an Applied Biosystems model 3730XL automated DNA sequencing instrument. The 18S rRNA sequences of the two yeast isolates, namely Pichia kudriavizvii and Hanseniaspora guillermondii (Y16 and Y26), were subsequently submitted to GenBank, where they were assigned accession numbers OL621856 and OL621857, respectively.

2.3.2 Sequence analysis of 18S rRNA gene and phylogenetic analysis

The 18S rRNA sequences of the two yeast isolates were provided to GenBank for analysis. Comparison was made between these sequences and the 18S rRNA gene sequences of Pichia kudriavizvii and Hanseniaspora guillermondii, which are available in the GenBank database. This analysis was carried out using the National Center for Biotechnology Information (NCBI) in Bethesda, MD, USA [25]. The alignment of the sequences with reference sequences from the NCBI database was performed using the MEGA X software. Subsequently, the phylogenetic dendrogram was constructed using the MEGA X program, and the unweighted pair group method with arithmetic mean (UPGMA) was employed to generate the phylogenetic tree [26].

3 Results and discussion

3.1 Isolation and morphological characteristics

A comprehensive investigation was conducted on a total of 42 yeast isolates for the purpose of evaluating their morphological characteristics and enzymatic activity. The yeast colonies underwent meticulous examination to document their morphological traits, which encompassed their shape (both circular and irregular), color (including white, creamy, and reddish brown), surface texture (both smooth and rough), and elevation (which ranged from raised and convex to umbonate). The yeast cells were carefully scrutinized and were found to possess oval, elongated, and spherical shapes, as indicated in Table 1 and Figs. 1 and 2. These findings align with the observations made by Yavad and Tiwari regarding the macroscopic characteristics of Saccharomyces, in which they noted the yeast’s smooth surface, circular margin, and yellowish color [27]. Based on macroscopic observations, the morphological characteristics of yeast colonies were determined to be white to creamy in color, with butyrous textures and dull surfaces [28]. The colored macroscopic morphology of the yeast was described as cream, with entire dull shapes and convex surfaces [29]. Furthermore, a motility test was conducted, and the results revealed that some of the isolates exhibited motility, while others were non-motile, as depicted in Table 1.

Table 1 Isolation and morphological characteristics of yeast isolates
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Fig. 1
figure 1

yeast isolate colonies on YEPD medium

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Fig. 2
figure 2

Gram staining of different yeast isolates cells

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3.2 Screening of enzymolytic activity

Forty-two isolates were examined to assess their capacity for the production of the enzymes pectinase, amylase, urease, lipase, protease, and β-galactosidase. Following the incubation period, it was determined that 30 out of the 42 isolates demonstrated the ability to generate hydrolytic enzymes, as outlined in Table 2. Among these enzymes, amylase, pectinase, lipase, urease, β-galactosidase, and protease were produced by 3, 13, 10, 13, 11, and 10 isolates, respectively.

Table 2 Enzymolytic activity of yeast isolates
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The findings revealed that the yeast isolates predominantly produced pectinase, protease, and urease enzymes, while amylase enzyme production was observed in fewer isolates, as indicated in Table 2. Two isolates concurrently produced amylase, pectinase, lipase, protease, and urease enzymes, while two other isolates generated lactase, pectinase, and lipase enzymes simultaneously. Additionally, seven isolates demonstrated simultaneous production of lipase and pectinase, as demonstrated in Table 3. In order to assess β-galactosidase activity at a basic level, certain isolates underwent a pH change from 6.0 to 3.5, 5, 8, and 9 pH. This resulted in the trapping of gas in the Durham tube, and a change in the color of the medium from orange to yellow and red, as depicted in Fig. 3.

Table 3 Multi-enzyme production by yeast isolates
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Fig. 3
figure 3

Screening for enzymolytic activity of different yeast isolate

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The outcomes in Table 4 manifest clear discrepancies in the levels of residual reducing sugars, signifying that yeast consumed lactose as the sole carbon source depending on the incubation period. The incubation period was a significant factor in the secretion of β- galactosidase enzyme. All isolates displayed superior lactose consumption after 5 days compared to 3 and 4 days. Isolate Y20 was identified as the most proficient in consuming lactose as the sole carbon source. The production of multiple enzymes from microbial sources is viewed as a valuable substitute for commercial enzymes, particularly in the breakdown of food waste and agro-industrial by-products [30, 31].

Table 4 Determination of residual reducing sugars
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The findings demonstrated that two yeast isolates, Y16 and Y26, possessed the ability to produce multiple enzymes and were consequently chosen for molecular identification. These outcomes were in agreement with previous research indicating that the genus Hanseniaspora is among the most prevalent non-Saccharomyces yeasts found on the surface of intact grapes, and are also recognized as one of the principal producers of glycolytic and other enzymatic activities [32, 33].

The results presented in Table 4 reveal distinct inconsistencies in the levels of residual reducing sugars, suggesting that yeast consumed lactose as the exclusive carbon source depending on the duration of incubation. The duration of incubation proved to be a significant determinant in the secretion of the β-galactosidase enzyme. After 5 days, all isolates exhibited superior lactose consumption compared to 3 and 4 days. Among the isolates, Y20 demonstrated the highest proficiency in utilizing lactose as the sole carbon source. The utilization of various enzymes derived from microbial sources is commonly regarded as a valuable alternative to commercial enzymes, particularly in the degradation of food waste and agro-industrial by-products [30, 31].

The findings of this study unequivocally demonstrated that two yeast isolates, namely Y16 and Y26, possessed the capability to generate multiple enzymes and were subsequently selected for molecular identification. These findings align with previous research, which has consistently indicated that the genus Hanseniaspora is one of the most prevalent non-Saccharomyces yeasts present on the surface of intact grapes. Additionally, it is recognized as a major producer of glycolytic and other enzymatic activities [32, 33].

3.3 Molecular characterization and identification of yeast isolates

The 18S rRNA gene, due to its highly conserved nature within species, is frequently employed as a molecular marker for biodiversity research, with similarities reaching nearly 100%. By utilizing universal forward and reverse primers specific to the 18S rDNA, the ribosomal DNA was amplified through the application of PCR, resulting in a 600 bp product (Fig. 4). The variability present within the amplified regions was subsequently investigated through the utilization of phylogenetic analysis. The amplified PCR product was visualized through electrophoresis on a 2% agarose gel and observed using a UV transilluminator.

Fig. 4
figure 4

Agarose gel electrophoresis after 18 s rDNA-PCR amplification of two yeast isolates. Lane Y16 represents Pichia kudriavizvii (OL621856) and Lane Y26 represents Hanseniaspora guillermondii (OL621857). Lane M represents the molecular size marker (100 bp leader)

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3.4 Phylogenetic analyses of the rDNA sequences

The outcomes of the phylogenetic analysis demonstrate that the isolates pertain to two distinct genera, namely Pichia and Hanseniaspora. By comparing the amplified PCR product to sequences stored in GenBank (http://www.ncbi.nlm.nih.gov), the isolates were identified and found to bear a close resemblance to known strains of P. kudriavizvii and H. guillermondii, as depicted in Table 5. The similarity index employed in the cluster analysis also confirms the affinity between the isolates and known strains.

Table 5 Identification of yeast isolates on the basis of 18 s rDNA gene sequence similarity
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The phylogenetic tree reveals the segregation of the isolates into two clusters, each corresponding to a genus, with a marked similarity to known strains, as illustrated in Fig. 5. The discoveries made in this study present fresh perspectives on the diversity of yeast species present in the collected samples, while simultaneously highlighting the effectiveness of utilizing 18S rRNA gene sequences for yeast identification. The outcomes of the cluster analysis signal a high level of similarity between the yeast isolates and specific species of yeasts that were previously identified. Specifically, isolate Y16 with accession number |OL621856| demonstrates a similarity index of 99.8% with Pichia kudriavizvii strains |KY104596|, |MN310532|, and |KY985267|. Similarly, isolate Y26 with accession number |OL621857| exhibits a similarity index of 99.86% with Hanseniaspora guillermondii strains |KY103525| and |EF449523|, as delineated in Tables 6 and 7. In conclusion, the results of this study demonstrate the close relationship between the yeast isolates and these specified yeast species.

Fig. 5
figure 5

Phylogenetic analysis of yeast isolates based on ITS region of geneomic rDNA gene showing the relationship between our two isolates and 10 representative strains. Evolutionary analyses were conducted in MEGA X. Rooted phylogentic tree (UPGMA)

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Table 6 Genetic similarity percentage of 5 strains of Pichia kudriavizvii and isolate Y16 with accession number OL621856 based on ITS region of genomic 18 s rDNA gene
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Table 7 Genetic similarity percentage of 5 strains of Hanseniaspora guillermondii and isolate Y26 with accession number OL621857 based on ITS region of genomic 18 s rDNA gene
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The results obtained validate the notion that both Pichia kudriavizvii and Hanseniaspora guillermondii isolates possess a substantial capacity for the synthesis of numerous enzymes. The capacity of yeasts to synthesize hydrolytic enzymes for pectin, protein, cellulose, and starch were assessed through the utilization of plate assays. In comparison with P. fermentans, Candida railenensis, C. xylopsoci, and Wickerhamomyces anomalus, H. uvarum and P. kudriavzevii demonstrated elevated activities in terms of pectinase, amylase, cellulase, and protease [34].

4 Conclusion

The findings derived from this investigation demonstrate that, out of a total of 42 yeast isolates, enzymolytic activity was observed in 30 isolates. Furthermore, it was observed that seven yeast isolates exhibited the capability of producing multiple enzymes, thus suggesting their potential for application in industrial enzyme production and environmentally sustainable industries.

Molecular identification techniques revealed that two specific isolates, namely Y 16 and Y 26, possessed the ability to produce multiple enzymes. Specifically, isolate Y 16 was identified as Pichia kudriavizvii-OL621856, whereas isolate Y 26 was identified as Hanseniaspora guillermondii-OL621857 strain.

The acquisition of these two yeast isolates is of significant value due to their capacity to generate a diverse array of secure enzymes, which find utility in various fields such as medicine, agriculture, and the food industry. Moreover, the rapid growth rate of these isolates, combined with favorable developmental conditions, facilitates the efficient production of a substantial quantity of secure enzymes within a short period of time and at a reduced cost. As a result, it is highly recommended that future research be conducted to investigate the genes responsible for the production of these enzymes, elucidate their sequences, and explore the potential of inducing mutations that enhance the efficiency of enzyme production.