Exploring protein derivative profiles in cheese whey through native Candida tropicalis fermentation

ABSTRACT

Native yeasts produce most peptides and amino acids from cheese whey fermentation. The research aimed to determine the protein, peptides, and amino acid profiles regarding whey fermentation by spontaneous fermentation and re-inoculation by native yeasts as a basis for determining potential yeasts in amino acid production. The selected native yeasts were identified through genome-based analysis on the DNA base sequence of the Internal Transcribed Spacer (ITS) region. The molecular identification shows that isolate CT1 is 95.62% comparable to Candida tropicalis strain VIT-NN02, CT2 is 98.85% same to stain NB5Y, and CT3 is 99.42% similar to GT 6. Whey fermentation was conducted by inoculating 3% each native C. tropicalis and without inoculation for spontaneous fermentation, then incubated for 48 hours at ambient temperature (26 ± 2°C). Unlike the other samples, the spontaneously fermented mozzarella whey was incubated without inoculation after cheese separation. After 48 hours, fermented whey was kept at 4°C for examination. The Kjeldahl method evaluated protein concentration, Folin-Ciocalteau determined peptide levels, and HPLC determined amino acid composition. C.tropicalis was identified as cheese whey native yeasts, and all treatments decreased protein and peptides while increased the total amino acid includes every single amino acid value except histidine during isolate CT1 whey fermentation. Candida tropicalis CT2 shown as potential isolate for the amino acid production from whey, with the protein contents of 0.846 ± 0.151%, protein decreases of 0.384 ± 0.186, peptide levels of 264.75 ± 40.40ppm, peptide decreases of 99.87 ± 37.96 and the highest total amino acid of 6149.56ppm.

KEYWORDS:

• Amino acid
• fermentation
• mozzarella cheese whey
• peptides

Introduction

Fermentation of whey can produce a variety of new high-value products, such as amino acids and peptides. The nutritional content of whey—4%-5% lactose, 0.6%-1% protein, 0.4%-0.9% minerals and vitamins — supports the growth of microorganisms, allowing whey to be fermented.^[1] Fermentation outperforms other foods processing methods and enriches products with vitamins, amino acids, and proteins and releases human-beneficial components.^[2] Amino acids and peptides are generally formed due to the metabolic activity of microorganisms that can break down protein molecules on the substrate into simpler molecules, namely amino acids, and peptides. Therefore, the formation of amino acids and peptides during fermentation is strongly influenced by the proteolytic activity of the microorganisms used. Amino acids and peptides are among the compounds or metabolites that can be produced by whey fermentation. Metabolites such as amino acids and peptides provide numerous advantages for the food industry and human health, including cysteine as a dough conditioner, glutamic acid as a flavoring agent, and phenylalanine as a precursor to aspartame.^[3] The immune system, neurology, antioxidant response, protein synthesis, reproduction, and growth are all affected by essential and non-essential amino acids.^[4] There are also peptides that shows antimicrobial, antioxidant, antihypertensive, immunomodulatory, and anti-diabetic activities.^[5]

Microorganisms produce amino acids and peptides through fermentation, which can result in metabolites, simple acids, alcohols, or carbon dioxide. Biotransformation reactions, such as glycol group residue removal, can also produce health-beneficial compounds.^[6] Using native yeast during whey fermentation can enhance the release and retention of bioactive peptides, potentially increasing the nutritional and therapeutic benefits of protein derivatives.^[7,8] However, using native yeast strains instead of chosen or commercial strains can result in unpredictable and diverse fermentation processes, resulting in protein derivatives with varying content, flavor, and quality. Unfavorable microorganisms may impact fermentation and protein quality, and native yeasts can increase fermentation time and production costs.^[9]

Native yeasts were found naturally ferment whey then produce amino acids and peptides.^[10–12] Candida lambica, Candida parapsilosis, Candida rugosa, Debaromyces hansenii, Kluyveromyces lactis, Kodamaea ohmeri, Torulaspora delbrueckii, Zygosaccharomyces rouxii, Candida ethanolides, Candida zelbidanus, Pichia coccus pseudolambica, Pichia farinosa, Candida mogii, Candida intermedia, Saccharomyces cerevisiae, Kluyveromyces marxianus, and Kluyveromyces marxianus, Clavispora lusitaniae, Galactomyces geotrichum, Candida parapsilosis, Cryptococcus albidus, Kodamaea ohmeri, Zygosaccharomyces rouxii, Torulaspora delbrueckii, Debaromyces hansenii, W. pararugosa and Candida intermedia are the yeasts that have been isolated from whey which shown the proteolytic activity.^{[10,11,13,14]}

Protease type affects yeast’s amino acid composition, with proteins exhibiting specificity and possessing multiple levels of determination. Candida tropicalis, found in cheese and their derivatives, exhibits proteolytic activities through SAPT1 genes, encoding secreted aspartyl protease.^[15,16] Candida tropicalis produces aspartyl and serine proteases that can hydrolyze various proteins, including bovine serum albumin, human serum albumin, laminin, and fibrinonectin.^[17] However, there are still pros and cons regarding C.tropicalis proteases activities, that all C. tropicalis isolates showed higher enzymatic production than C. albicans, nevertheless it is contradictory to most of the studies which suggest higher proteinase activity in C. albicans than in C. tropicalis.^[18] Based on the explanation above, it is important to determine the roles of native microorganisms especially yeasts toward the protein derivatives of mozzarella cheese whey. The research aimed to identify the natives Candida tropicalis isolated from cheese whey and their capacity in resulting peptides and amino acids. This research will also highlight fermentation’s potential as an alternative method for applying cheese whey, which reduces environmental pollution and benefits the food industry.

Materials and methods

The research was done through explorative methodology, the descriptive analysis was done to identify and determine the native yeasts strain isolated from mozzarella cheese whey taken from KPBS Pangalengan and how they affect the protein derivatives insides. After the native yeasts were isolated and identified using molecular approach, the activities in resulting protein derivatives were analyzed. The protein and peptides data taken based on completely randomized design with six replications, then the data analyzed with ANOVA and followed by Tukey test which processed with Origin Pro 2021.

Species identification of native Candida tropicalis

Candida tropicalis isolation was carried out by taking 1 ml of mozzarella cheese whey, carrying out a series of dilutions, and then inoculating it on solid media. The media commonly used for yeast growth are YMA (Yeast Mold Agar, Oxoid, UK) and 10 ppm amoxicillin (Kimia Farma, Indonesia) to prevent the growth of contaminant bacteria. The yeast identification was carried out by genome-based analysis on the DNA base sequence of the Internal Transcribed Spacer (ITS) region. The primers used were ITS1 as a reverse primer and ITS4 as a forward primer. ITS1 primer sequence is (5”-TCCGTAGGTGAACCTGCGG-3‘) and ITS4 primer is (5’- TCCTCCGCTTATTGATATGC-3”).^[19] The PCR conditions: initial denaturation at 95°C for 1.5 min; 30 cycles of denaturing at 94°C for 2 min; annealing at 60°C for 1 min, an extension at 72°C for 2.5 min; and a final extension step of 5 min at 72°C. The products were electrophoresed in 1.5% agarose gel and visualized.^[20] The analysis of the sequencing results begins with contigting the base sequences of the ITS regional coding DNA using the Contig Assembly Program (CAP) application which is integrated with the MEGA X software. The contiguous sequences are aligned with the sequence data contained in the Genbank database using Basic Local Alignment Search. Tool (BLAST) integrated with NCBI. Alignment results are used for species identification.

Reconstructing the cheese whey native yeasts phylogenetic tree used the neighbor-joining (NJ) method. The NJ method selects sequences that, when combined, will provide the best estimate of the closest branch length and reflect the real distance between the sequences.^[21] The phylogenetic tree reconstruction used companion sequences from the NCBI GenBank, which were chosen because they were considered to have the highest relationship.

Identification of fermented cheese whey protein derivatives

The main research conducted was mozzarella whey fermentation which was carried out based on Isfari Dinika et al. (2019) methodology with modifications.^[11] The fermentation was carried out spontaneously and by inoculation of the native yeasts isolated from the mozzarella whey. Prior to fermentation, native yeasts were enriched on modified YMA media and then incubated at temperature of 26 ± 2°C for 48 hours. After incubation, the colony from the cultured YMA was taken, then inoculated to the Yeast Mold Broth (YMB) and standardized using McFarland 0.5 to obtain an inoculum with a yeast count of 1.5 × 10⁸ CFU/ml.

Whey fermentation begins with mozzarella whey pasteurization at 80°C for 15 minutes. A total of 3% inoculum (v/v) standardized with McFarland 0.5 was inoculated into the mozzarella whey at room temperature. The mozzarella whey was incubated for 48 hours at temperature of 26 ± 2°C. For spontaneously fermented mozzarella whey, it was not pasteurized and inoculated, it is immediately incubated after cheese separation process for 48 hours, the fermented whey was then stored at 4°C.

The samples were further analyzed for protein content using the AOAC Official Method 998.05 Non casein Nitrogen Content of Milk Kjeldahl Method.^[22] 15.00 grams of K₂SO₄ were added, 1 milliliter of CuSO₄.5 H₂O, and 8–10 boiling stones to the Kjeldahl flask. The sample (5 ± 0.1 ml) is placed in the Kjeldahl flask at 38°C. Boil 25 ml H₂SO₄ for 20 minutes until white steam forms. The sample is cooked until transparent with a faint blue-green tint. After clearing, heat the solution for 1–1.5 hours. After cooling to room temperature, 300 ml H₂O is added. Separately, add 50 ml H₃BO₃ and methyl red indicator to the Erlenmeyer flask and place it on the condenser end until the tip is submerged. Add 75 ml of 50% NaOH to the cooled sample solution and attach the flask to the distillation bulb in the condenser. Stir the pumpkin’s contents. Distillation to 150 ml distillate and 200 ml total volume. Remove the H 3 BO 3 Erlenmeyer flask and let the condenser drip the leftover liquid. until pink. Record HCl usage.

Peptide levels were analyzed using the Folin-Ciocalteau method,^[23] then compared to obtain the different protein content and peptide content. An aliquot of 15.5 ml of sample was taken and 12% (w/v) TCA was added to the same volume (15.5 ml), then continued with 15-minute centrifugation at 18,000 rpm. The supernatant was filtered using a 0.45 μm acrodisc, then 1 ml of supernatant was combined with 5 ml of 2% sodium carbonate, 1% copper sulfate, and 2% potassium tartrate in 0.1 N NaOH. Darkly incubating the mixture for 10 minutes. As much as 0.5 ml of Folin reagent which has been diluted with distilled water in a ratio of 1:1 (prepared when it will be used) is added to the mixture and then incubated for 30 minutes in a dark room. Absorbance was measured using a UV Vis Spectrophotometer at 590 nm.

Amino acid composition was identified using the HPLC method.^[11] The screw tube inserted with 6 mg of protein and 2 mL of 6 N HCl. The screw tube containing the sample solution is filled with nitrogen gas for 0.5–1 minutes and closed. For hydrolysis, the closed tube was baked at 110°C for 24 hours. After room temperature, the hydrolyzed sample was quantitatively transferred to the rotary evaporator flask. Tube rinsed with aquadest 2–3 times, then the rinsing solutions mixed into the rotary evaporator flask and the samples dried. Dried samples were added by 0.01 N HCl until 10 mL, then dissolve hydrolyzed sample were filtered with milipore paper and Potassium Borate Buffer pH 10.4 were added (1:1). Later on, 5 μl of samples were mixed with 25 μl of OPA (o-phthalaldehyd) reagent then added to a clean vial and leave for 1 minute to finish the derivatization. The 5 μl samples were injected into HPLC column then wait until all amino acids separates approximately 25 minutes.

Results

Species identification of native C.tropicalis isolated from mozzarella whey

Three native C.tropicalis were isolated from mozzarella whey, named CT1, CT2, and CT3 are represented in Table 1. The results showed that the CT1 isolate was 95.62% identical to the Candida tropicalis strain VIT-NN02 sequence registered on GenBank with access number MG309787.1.^[24] Meanwhile, the CT2 isolate was 98.85% identical to the Candida tropicalis strain NB5Y registered on GenBank with access number MT102793.1.^[25] Meanwhile, the CT3 isolate was 99.42% identical to the Candida tropicalis isolate GT_6 registered with GenBank with the access number MT228993.1.^[26] In general, identifying an isolate can be determined from the percent identity value obtained through BLAST analysis. If the homologous DNA content ranges from 60% to 100%, it is considered an identical species 20–60% is considered a closely related species, whereas if < 20% is considered a different species.^[27]

Table 1. Natives Yeasts Species Identification based on BLAST.

Parameter	Isolate CT1	Isolate CT2	Isolate CT3
Description	Candida tropicalis strain VIT-NN02	Candida tropicalis strain NB5Y	Candida tropicalis isolate GT_6
Max score	837	928	946
Total score	837	928	946
Query cover	99%	99%	99%
E Value	0.0	0.0	0.0
Per. Identity	95.62%	98.85	99.42%
Accession	MG309787.1	MT102793.1	MT228993.1

The reconstruction of the phylogenetic tree, as shown in Figure 1, revealed that the CT1 isolate sequence was in a monophyletic group with Candida tropicalis strain VIT-NN02, indicating that they were very closely related species or believed to have originated from the same genetic source. Consequently, they still retained the original characteristics of their ancestors. Similarly, the CT3 isolate was found to be closely related to the Candida tropicalis isolate GT 6. Additionally, the CT3 isolate exhibited a close relationship with the CT2 isolate, suggesting that these two isolates shared a common ancestor. In contrast, the CT2 isolate showed a low relationship with its closest species, Candida tropicalis strain NB5Y, as it belonged to a different branch.

Figure 1. Phylogenetic Tree of Natives C.tropicalis Isolated from Mozzarella Whey.

Protein and peptide contents of fermented whey

The protein and peptide contents analysis results of all the treatments are presented in Figure 2 and Table 2. The protein contents of whey were significantly decrease, however as shown on the Tukey test results, the decreases between all of treatments were not significant. The protein levels of CT2 isolate was significantly different with CT3 isolate, however both of them were not significantly different toward the spontaneous fermentation and the CT1 isolate. Meanwhile the peptides levels were significantly different, and the peptide decreases was also significant.

Figure 2. Protein levels (a) the decrease of Protein (b) Peptides levels (c) and the decrease of Peptides (d) of Fermented Whey.

Table 2. Protein and Peptide Contents in Fermented Whey Samples.

Parameter	Whey*	SF**	Isolate CT1	Isolate CT2	Isolate CT3
Protein (%)	1.230^a ± 0.244	0.797^bc ± 0.047	0.717^bc ± 0.031	0.846^b ± 0.151	0.609^c ± 0.001
Protein decrease against Whey (%)	-	0.433^a ± 0.265	0.514^a ± 0.233	0.384^a ± 0.186	0.621^a ± 0.243
Peptide (ppm)	364.63^a ± 4.03	214.97^c ± 13.42	290.93^b ± 47.95	264.75^bc ± 40.40	248.97^bc ± 41.38
Peptide decrease against Whey (ppm)	-	149.65^a ± 11.22	73.69^b ± 44.05	99.87^ab ± 37.96	115.66^ab ± 38.18

*Not Fermented; **Spontaneously Fermented; Different letters indicate statistically significant differences (p ≤ 0.05).

Amino acid profiles of fermented whey

Table 3 shows the highest to lowest total amino acid levels, respectively, whey samples fermented by C. tropicalis CT2, C. tropicalis CT3, spontaneous fermentation, and whey fermented by C. tropicalis CT1. Amino acid levels in fermented whey increased on average except for histidine whey fermented C. tropicalis CT1, which showed a decrease of 15.08% against the initial whey. The highest increase in amino acid levels occurred in glycine whey fermented C. tropicalis CT2 with an increase of up to 4770.49%.

Table 3. Amino acid profiles of fermented whey by native C.tropicalis.

	Whey*	SF**	Isolate CT1	Isolate CT2	Isolate CT3
Amino acid	(ppm)
Glycine	64.90	1771.49	1471.51	3096.05	2293.09
Aspartate	158.21	216.17	186.65	376.33	287.42
Glutamate	88.92	401.29	319.02	649.38	473.48
Serine	20.25	111.94	88.03	180.55	129.95
Histidine	38.44	39.23	32.65	64.28	44.95
Threonine	58.64	133.57	107.51	213.83	156.05
Arginine	21.32	65.84	53.24	122.96	81.19
Alanine	40.21	94.25	80.23	168.52	130.35
Tyrosine	16.51	55.83	43.94	88.30	61.91
Methionine	9.71	43.00	28.90	58.86	44.44
Valine	42.58	130.92	102.04	201.67	145.77
Phenylalanine	17.67	77.75	59.58	122.38	83.70
L-Isoleucine	58.30	140.36	100.07	203.72	153.75
Leucine	39.74	220.64	165.65	335.44	242.82
Lycine	127.94	171.73	135.33	267.32	201.47
Total	804.35	3673.99	2974.35	6149.56	4530.35

*Not Fermented; **Spontaneously Fermented.

Discussion

Species identification of native C.tropicalis isolated from mozzarella whey

Based on the result (Table 1), the total score and maximum score of isolates CT1, CT2, and CT3 were 837, 928, and 946, respectively. The total score indicates the total value of base pairs, while the maximum score indicates the value of the similarity of base pairs between the query and subject. The higher the maximum score, the higher the level of identification. On the other hand, the query cover value of the three sequences is 99%, meaning that the query coverage indicates the percentage of nucleotide samples used in the BLAST analysis.

The results of BLAST analysis show that the percentage of identification is high, which is indicated by the e-value of 0.0. The e-evalue is an estimated value that provides a statistically significant measure of both sequences. The lower the e-value, the higher the level of homology between the two sequences.^[28] According to Claverie and Notredame,^[29] if the e-value is 0.0 then the sequences have a very high identity, and based on the results, the isolate is similar to C.tropicalis. Candida tropicalis has been found in dairy-based products, cheese, and also in cheese whey, such as mozzarella cheese whey.^[10]

Phylogenetic reconstruction of isolates CT1, CT2, and CT3 placed Candida sp. EBY21 as an outgroup (Figure 1). According to Hidayat and Adi,^[30] outgroup groups are needed and form a tree that is divided into apomorphic and plesiomorphic characters. The phylogenetic tree of isolates CT1, CT2, and CT3 was also tested statistically by the bootstrap test with 1000 replications. The bootstrap test of 100–1000 replications is effectively used to estimate the confidence level of a phylogenetic tree.^[31] The larger the bootstrap replication, the higher the truth level of the reconstructed tree topology based on the distribution of characters in the data, which is strongly influenced by random effects.^[32]

The CT1 and CT3 isolates are located at the branching with bootstrap 100, and the CT2 isolate is located at the branching with the bootstrap value 87. These results indicate that the sequence grouping in each phylogenetic tree formed has a high level of topological confidence because it has a bootstrap value above 70.^[33] The value of the scale bar on the phylogenetic tree of native yeast isolates CT1, CT2, and CT3 is 0.01, which indicates a genetic distance with a nucleotide change of 1 time every 100 bp.^[34] The value of the genetic distance can describe the genetic relationship. A low genetic distance describes a close genetic relationship if two or more species have a genetic distance value of less than 0.03, indicating that the species has genetic closeness.^[35,36]

Yeasts have been found as one of the major microbial groups in some cheese kinds after studies on the presence of yeasts in many cheese variations, including whey cheeses.^[37] Due to their capacity to use the whey protein content for fermentation, Candida tropicalis are present in cheese whey as a typical microbiota.^[10] A considerable portion of milk proteins can be found in whey, a byproduct of the cheese-making process. Through fermentation with Candida tropicalis, the protein content of cheese whey may be transformed into peptides and amino acids.^[11] This yeast species participates in peptide and amino acid modifications during the fermentation of cheese whey, which may help explain why it is a normal component of the microbiota.^[10] Given their capacity to ferment cheese whey, Candida tropicalis yeasts may be present if whey spontaneously ferments.

Protein and peptide contents of fermented whey

Based on the results on Table 2, whey that inoculated with C. tropicalis CT3 had the lowest protein contents which shows highest protein decreases and C. tropicalis CT2 shown the highest protein contents with the lowest protein decreases against the initial whey. However, the protein decreases of all of the treatments were not significant. Meanwhile the highest peptides contents shown by the C. tropicalis CT1 treatment which mean the lowest decreases of peptides, and the lowest peptides contents resulted by spontaneous fermented whey, which mean the highest decreases of peptides. Peptide levels of spontaneous fermented whey was significantly different with the initial whey and whey that inoculated by isolate CT1, nevertheless the SF and CT1 was not significant toward isolate CT2 and CT3. Also for the peptide decreases that significant different between SF and CT1, while both of them was non-significant against CT2 and CT3.

The protein concentration tends to decreased because yeasts protein-metabolizing activity is increased. The yeast’s requirement for nitrogen drives proteolytic activity, which lowers protein and nitrogen levels.^[38] During fermentation, yeast releases proteases that break proteins into peptides and amino acids. This breakdown allows yeast cells hydrolyzes peptides and amino acids that provide nitrogen for yeast growth.^[9] The amount of protein content in whey depends on the intensity of the proteolytic activity of each yeast which may vary. Substrates rich in protein, such as whey, can trigger an increase in yeast proteolytic activity so that more protein can be hydrolyzed into amino acids.^[39]

The results also shown the higher % decrease of proteins resulting low peptides decreases as found in CT1, however in other treatments also found that the higher % decrease of proteins also resulting in high decreases of peptides as shown in SF, CT2 and CT3. Pre heat treated whey may have selected microorganisms such Candida spp. and lactic acid bacteria that could grow without thermolabile nutrients, which contribute to proteolysis and flavor development.^[40] It can be surmised that during fermentation processes, proteins undergo degradation, which involves the breakdown of proteins into peptides and eventually into amino acids.^[41] This degradation can occur through enzymatic hydrolysis or non-enzymatic breakdown.^[42] The enzymatic hydrolysis through the discharge of peptidases into the environment may be a significant adaptative component for the yeast’s life cycle, which is possible resulting continue the protein degradation process from peptides into amino acid.

C. tropicalis could produce peptides up to 1680 ± 230 ppm with proteolytic activity of 1450–1910 ppm, which in the first 24 hours yielded 10.42 ppm peptide.^[11,43] Spontaneously fermented whey had the most peptides decreases, it is plausible has more microorganisms especially lactic acid bacteria. Whey contains several thermophilic lactic acid bacteria such as Streptococcus sp., E. durans, Streptococcus gallolyticus macedonicus, Aerococcus viridans, and E. faecium, along with yeast, are also suspected to be responsible for the spontaneous fermentation of whey.^[44] Co-culturing microorganisms accelerates proteolytic activity to increase peptides decreases.^[45] Due to microbial synergy during fermentation, the presence of other microorganisms can give a signal as a trigger for peptides-hydrolyzing microorganisms.^[46] The synergy shows natives Candida spp. excretes at least one protease that hydrolyzes α-lactalbumin, while Lb. paracasei acidifies medium to activate proteases that resulting in peptides release together with pH decreases.^[40] Meanwhile, whey fermented by the yeasts did not have the same diversity of microorganisms as spontaneously fermented whey. This is due to the sterilization and inoculation process of yeasts fermented whey, which can suppress the life of undesirable microorganisms.^[47]

Fermentation raised whey peptide levels through yeast proteolysis activities, however in some point yeasts also hydrolyzed the peptides into amino acids. Yeasts utilized the proteins in whey as a source of nitrogen for development and metabolism, through proteolytic enzymes or proteases production, which break down proteins into peptides.^[48] As fermentation progresses, yeasts can hydrolyze peptides into amino acids through peptidases action that cleaving the peptide bonds.^[11] The amino acids provide nutrients for yeast growth and contribute to the overall fermentation process.

Amino acid profiles of fermented whey

The results on Table 3 showed that the levels of each type of amino acid from fermented whey generally increase. The increase in amino acids in spontaneously fermented whey can be caused by yeast or lactic acid bacteria, although yeast is thought to dominate more than lactic acid bacteria. This refers to Ponomarova et al.. (2017), which states that lactic acid bacteria have more diverse amino acid requirements than yeasts, so they are more likely to consume existing amino acids rather than secrete them. Meanwhile, yeast can secrete various amino acids such as threonine, glutamate, serine, alanine, glycine, valine, and isoleucine.^[49,50] That is why the total amino acids were still higher on the treatments of isolate CT2 and CT3.

The difference in total amino acid content between whey that was naturally fermented and whey that was fermented by C. tropicalis was caused by the different microorganisms that were present during fermentation. More microorganisms, like lactic acid bacteria and yeasts, are involved in fermentation that happens on its own. During fermentation, the bacteria work together to break down proteins and make amino acids [64]. Lee et al.. (2014) said that fermentation with mixed cultures could boost the proteolytic system, causing more amino acids that the microorganisms need to live to be released.

The highest increase in amino acid type in all of the treatments was shown by glycine especially in isolate CT2 treatment. In general, glycine assumes as an urgent part in one carbon utilization, thus promoting cell division, and also fills in as the precursors of L-serine and cysteine which can be secreted by lactic acid bacteria, as well as yeast.^[51–54] Current proof shows that variety in the cysteine biosynthesis is influenced by intracellular glutathione production, in which glycine is involved.^[53,55]

In addition to glycine, the quantity of glutamate increased significantly in fermented whey. Yeast and lactic acid bacteria produce glutamate by deaminating glutamine with the enzyme glutaminase.^[56,57] Glutamate is utilized as an amino group donor in numerous biosynthetic reactions, where it plays a crucial function.^[58] Glutamate amino group transfer occurs in the synthesis of several amino acids, including aspartic, phenylalanine, valine, isoleucine, and leucine, which also exhibit a rise in spontaneous fermentation.^[59] Although glutamate is used in the production of the aforementioned amino acids, glutamate levels increase in spontaneously fermented whey. This indicates that the glutamate production rate is greater than the glutamate consumption rate [59]. The high output can be attributed to its products, which are derived from both lactic acid and yeasts.^[57]

The results also revealed a significant increase in leucine. Yeasts’ metabolic pathways for the production of leucine, isoleucine, and valine overlap, inhibiting the production of the three amino acids each other.^[60,61] The increase in isoleucine in fermented whey can result from the conversion of threonine.^[62] At various phases of isoleucine production, leucine, valine, and isoleucine itself inhibit its production. Meanwhile, 2-keto-isovalerate, a precursor of valine, is also a precursor of leucine.^[63] This may explain why leucine has increased significantly more than isoleucine and valine.

The production of aspartate, threonine, and isoleucine has pathways that are directly or indirectly related to yeast metabolism. Yeast can produce aspartate through glycolysis.^[56,57] Aspartate is a precursor of threonine, while threonine is a precursor of isoleucine.^[63,64] Meanwhile, lactic acid bacteria can also produce aspartate through the deamination of asparagine.^[57] Therefore, aspartate production by yeast and lactic acid bacteria can support the increase in the amino acids as shown on the fermented whey.

The results also shown significant increase of aspartate and lysisne by the activity of yeasts isolates in this study compared to the activity of mixed cultures in SF. Enzymes involved in the metabolism of aspartate are found in yeasts, including Candida tropicalis. Aspartate can be metabolized via a number of different mechanisms, including the aspartate oxidase and transamination pathways. Key metabolites that are involved in energy production and biosynthesis, such as oxaloacetate, fumarate, and α-ketoglutarate, are produced as a result of these processes.^[16] One of the way to hydrolysis of the amino acid lysine in Candida tropicalis is through the saccharopine pathway, which involves the conversion of lysine to saccharopine via lysine-ketoglutarate reductase that can catabolize lysine. Saccharopine undergoes further metabolism to become α-aminoadipate, which starts the TCA cycle.^[65]

Whey fermentation by C. tropicalis CT1 results in the lowest content of all amino acid types. It is suspected that C. tropicalis CT1 can’t use lactose as well as it could, it uses the galactose it already has and sets off a process called catabolite carbon derepression, that forced the available amino acids will work as a reductive substrate for the Krebs cycle.^[66,67] This will provide electrons for energy transfer by the mitochondria, which yeast needs to grow.^[67] Meanwhile, the isolate CT1 resulting in histidine as the only amino acid that experienced a decrease compared to the others. Amino acids can also undergo deamination or transformation into other compounds.^[68] Candida spp. rather introduces histidine degradation by means of aminotransferase Aro8 activity toward the aromatic amino acid.^[69] Non-albicans Candida has adjusted to utilize histidine as a sole nitrogen source and utilizes an aromatic aminotransferase rather than a histidinase for histidine usage.^[70] The growth of Candida spp. considerably slower with aromatic amino acids such as phenylalanine, tryptophan, tyrosine, and also histidine.^[71] Significantly, histidine upholds the development of N-Acetylcysteine (NAC) through Ehrlich metabolic pathways to utilize histidine as a potential nitrogen source.^[72]

The isolate C.tropicalis CT2 shown the potential in producing amino acid that resulting in the lowest decreases of protein with promising activities in peptide decreases which resulting highest increases in various amino acid in fermented whey. The results could because the yeasts have a hierarchy of preferences for certain nitrogen sources that are regulated through a mechanism called Nitrogen Catabolite Repression.^[73] This mechanism allows the prevention of nitrogen consumption from a less preferred source or amino acid when a preferred nitrogen source is available.^[74] This occurs through equal emphasis on those responsible for regulating the uptake and catabolism of preferred nitrogen sources.^[73,75] Amino acids such as aspartate, histidine, threonine, arginine, alanine, tyrosine, methionine, valine, leucine, and phenylalanine, in some non-Saccharomyces yeasts are indeed used as a source of nitrogen intermediates, are less preferred, and are not even assimilated.^[76]

Conclusion

Through BLAST analysis, the study identified Candida tropicalis as the dominant yeast as native species isolated from mozzarella cheese whey, with high levels of similarity (>95%) showed by the isolates CT1, CT2, and CT3. The fermentation process led to a decrease in protein content and an increase in peptide levels in whey, which can be attributed to the proteolytic activity of native microorganisms, including yeast. The study also found that spontaneously fermented whey exhibited the highest deccreases in peptides, indicating the presence of diverse microorganisms involved in the process. Furthermore, the study investigated that generally the amino acid was increased regarding all of the treatments of whey fermentation. there was an increase in protein and peptides while increasing the total amino acid except histidine during isolate CT1. Candida tropicalis CT2 shown as potential isolate for the amino acid production from whey, with the protein contents of 0.846 ± 0.151%, protein decreases of 0.384 ± 0.186, peptide levels of 264.75 ± 40.40ppm, peptide decreases of 99.87 ± 37.96 and the highest total amino acids of 6149.56ppm. It was observed that C.tropicalis CT2 resulted in highest total amino acid content compared to other treatments includes the spontaneous fermentation. This suggests that C.tropicalis CT2 is potential for amino acid production with high increases in glycine, glutamate, serine, arginine, methionine, phenylalanine, and leucine. All fermentation treatments showed an increase in total amino acid contents, including the increase of all amino acid except histidine at fermented whey by C. tropicalis CT1. This research can continue to be developed in the future with improvements in the methods carried out, both advanced molecular and other methods that are currently being developed. Apart from that, currently research is limited to the study of protein derivatives only, and in the future more in-depth studies can be carried out on other compound components.

Authors contributors

Conceptualization, methodology, data curation, analysis, writing, review, and editing, G.L.U.; data curation, analysis, writing, F.U., V.F.S.; writing and editing, S.N.; supervision, validation, Y.C.; and funding acquisition, R.L.B. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

The authors thank to Meli Puspita Sari for the contribution in improving the quality of the manuscript, the Laboratory of Food Microbiology, Department of Food Industrial Technology, Faculty Agroindustrial Technology, Universitas Padjadjaran, and the Directorate of Research, Community Services of Universitas Padjadjaran, who has funded the publication.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

Directorate of Research and Community Services, the Ministry of Research, Technology and Higher Education the Republic of Indonesia through the scheme of “Penelitian Dasar”.