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Full Research Article

Fermentation-induced Acidification and Structure Formation in Donkey Milk

N
N.K. Turganbaeva1,*
0000-0002-7620-9236
R
R. Sh. Elemanova2
J
J.N. Smanalieva2,3
M
M.M. Musulmanova2
1Kyrgyz-Turkish Manas University, Bishkek, Kyrgyz Republic.
2I. Razzakov Kyrgyz State Technical University, Bishkek, Kyrgyz Republic.
3Technical University Dresden, Dresden, Germany.

ABSTRACT

Background: The search for new physiologically functional ingredients has led to increased attention toward donkey milk, recognized as a promising raw material for various food products, including fermented ones. This study investigates the potential of donkey milk for fermented dairy production by examining curd formation during milk biotransformation with different starter cultures.

Methods: The study to evaluate acid-forming properties of donkey milk employed the following starter cultures: Lactobacillus acidophilus, Bifidobacterium subsp. lactis, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Additionally, bacterial concentrates of Lactobacillus plantarum and Propionibacterium freudenreichii were used. Rheological parameters were determined using oscillatory measurement.

Result: Industrial starter cultures exhibited high growth and biochemical activity in donkey milk. A consortium of lactic and propionic acid bacteria achieved optimal acidification and coagulation. For the first time, rheological analysis - including complex viscosity, storage and loss moduli - characterized donkey milk curd, confirming its suitability for fermented products. Results showed that specific culture combinations effectively regulate titratable acidity and curdling coefficients. These findings established the technological basis for a novel fermented milk-plant beverage, demonstrating the potential of donkey milk as a functional dairy base.

INTRODUCTION

In recent years, consumer interest in donkey milk has steadily increased and it is gaining popularity across various markets. This product is notable not only for its distinctive taste but also for its high content of biologically valuable components. Despite its favorable nutritional profile, donkey milk was previously considered a niche product with limited accessibility for the public. However, the growth of e-commerce has significantly increased its availability, as many producers now market donkey milk through online platforms that offer international delivery. The growing consumer loyalty toward donkey milk reflects a broader global trend toward the consumption of natural and functional food products. In the Kyrgyz Republic, there is also a noticeable rise in interest in donkey milk as a source of bioactive compounds. Although religious dietary restrictions classify donkey milk as haram, its consumption for wellness purposes is increasing, often accompanied by positive anecdotal reports of its health benefits.
       
It is well established that human, donkey and mare’s milk are characterized by high biological and nutritional value, owing to a well-balanced amino acid composition and elevated lactose content (Felicita et al., 2014, Madhusudan et al., 2020; Altomonte et al., 2019; Giosue et al., 2008; Wszołek et al., 2014; Bhardwaj et al., 2019). Furthermore, these types of milk are rich in α-albumin, with an average particle size not exceeding 15-20 nm (Zhang et al., 2020; Ostroumova, 2004). The proportion of whey proteins in donkey and mare’s milk accounts for 35-50% of total protein content (Felicita et al., 2014; Madhusudan et al., 2020; Giosue et al., 2008; Wszołek et al., 2014), which categorizes them, along with human milk, as albuminous types. The low casein content promotes the formation of fine, soft curds during gastric coagulation, which are easily digestible and suitable for infant nutrition (Gorbatova, 2010). Moreover, the significantly reduced casein concentration in donkey milk contributes to its hypoallergenic properties, making it a valuable alternative for individuals with cow’s milk protein allergy (Cunsolo et al., 2017; Vincenzetti et al., 2014; Polidori et al., 2015; Prasad, 2020; Nayak, 2020).
       
The high levels of lactose and lysozyme in donkey milk open up opportunities for the development of new types of fermented dairy products (Cavalcanti et al., 2021; Vincenzetti et al., 2017; Dambrosio et al., 2023; Perna et al., 2015). Fermented dairy products are considered an optimal dietary option for individuals with lactose intolerance, as the fermentation process breaks down lactose into lactic acid, which positively influences the secretory function of the gastrointestinal tract. Lactose is the main substrate for acid production during fermentation, converted into lactic acid by the enzymatic activity of lactic acid bacteria in starter cultures.
       
The use of probiotic starter cultures is driven by their well-documented health benefits, including immunomodulatory, antimicrobial and antioxidant properties (Cousin et al., 2012; Hashemi et al., 2023; Rosa et al., 2022). The potential of Streptococcus thermophilus and Bifidobacterium subsp. Lactis strains involved in folate biosynthesis were investigated and it was found that when they are cultivated together, folate concentrations in the final product can increase sixfold (Adriani, 2024). These microorganisms play a crucial role in enhancing the nutritional value of fermented products and supporting gastrointestinal health (Yang et al., 2019; Mantel et al., 2022). Starter cultures play a crucial role in shaping the organoleptic characteristics of fermented dairy products. Lactic acid, produced during fermentation, functions as a natural preservative, enhancing the freshness and microbiological safety of the final product. However, the behavior of lactic acid bacteria and other microorganisms in starter cultures when introduced into donkey milk from the Kyrgyz population remains largely unexplored. This formed the basis of our study. The findings provide valuable insight into the technological properties of donkey milk as a substrate that supports the growth of starter microflora and the associated accumulation of lactic acid, leading to casein coagulation and the formation of a fermented milk curd.

MATERIALS AND METHODS

The work was carried out in the Department of Food Production Technology at the Technological Institute of the Kyrgyz State Technical University named after I. Razzakov during 2022-2023.
       
Donkey milk samples were obtained from two clinically healthy, lactating Kyrgyz donkeys aged 5 years, kept on a farm belonging to the Karà-Zhygach state agricultural enterprise in the Chüy Valley of the Kyrgyz Republic, at an altitude of 800 meters above sea level.
       
The preparation of milk samples for fermentation involved mild pasteurization (75°C for 15 minutes) (Luo et al., 2019, Giacometti et al., 2016; Wang et al., 2022), which was selected due to the low thermal stability of this type of milk. Following heat treatment, the samples were cooled to the inoculation temperature of 35°C.
       
The physicochemical characteristics of donkey milk from Kyrgyzstan are presented in Table 1.

Table 1: Physical and chemical composition of donkey milk from Kyrgyzstan.


       
As a comparative control, ultra-pasteurized cow’s milk of the brand “Vesyolyy Molochnik” was used produced by “Bishkeksut”, with a fat content of 1.5%, protein 2.9% and carbohydrates 4.7%.
       
To investigate the acid-forming properties of the starter cultures, the following microorganisms were utilized: Lactobacillus acidophilus (probiotic), Bifidobacterium subsp. Lactis (probiotic), Streptococcus thermophilus (probiotic) “Lyofast SAB 439 A” and Lactobacillus delbrueckii subsp. bulgaricus (probiotic) “Lyofast SAB 438B” (BioChem, Italy), each containing not fewer than 1011 CFU/g. In addition, a bacterial concentrate of Lactobacillus plantarum (probiotic) with a cell concentration of at least 1010 CFU/g, including phage-resistant strains for prolonged use (BioChem, Italy), was employed. Also included was a Propionibacterium freudenreichii (probiotic) bacterial concentrate (BioChem, Italy) with a cell density of at least 3×1010 CFU/g. The amount of inoculated culture followed the manufacturer’s recommended dosage.
       
The optimal growth temperature range for these microorganisms was between 30 and 45°C.

The rate of acidification, expressed in °T per unit of time, was determined using Equation (1) (Topel, 2012; Elemanova et al., 2022).
 
             Is = TAC2 - TAC1                          ...(1)
 
Where,
TAC1- Titratable acidity at the beginning of the observation period (°T).
TAC2- Titratable acidity at the end of the observation period (°T).
       
The acidification coefficient, which reflects the intensity of acidification relative to the observed increase in titratable acidity, was calculated using Equation (2) (Topel, 2012; Elemanova et al., 2022).

 
Where,
ΔTAC2- The difference between the initial acidity and the acidity measured at the end of the observation period (°T).
ΔTAC1- The difference between the initial acidity and the acidity measured at the beginning of the observation period (°T).
 
Rheological measurements
 
Rheological properties, namely complex viscosity (η), storage modulus (G′) and loss modulus (G″), were assessed by oscillatory measurement on the MCR 302 rheometer (Anton Paar, RheoCompass, Austria) using a time-sweep test, following the procedure described by Smanalieva et al. (2021). Data acquisition and processing were carried out with RheoCompass software, version 1.30. Milk samples were inoculated with the combined starter cultures and, at 35°C, transferred to the rheometer’s measuring cup. Rheological measurements were conducted over an 8-hour period under the following oscillation settings: strain amplitude γ = 1 %, frequency f = 1 Hz, employing a concentric-cylinder geometry (CC27-SN26341).
       
The formulation of the functional beverage was optimized using a linear programming mathematical model via LINDO software (Lindo Systems, 2020). Based on the optimization results, the optimal ratio of donkey milk to carrot puree was determined to be 70:30 (v/v).
       
Statistical analysis was performed in SPSS 22 (SPSS Inc., Chicago, IL) using paired-sample t-tests with a 95% confidence interval; differences were considered statistically significant at p<0.05.

RESULTS AND DISCUSSION

Acid formation during donkey milk fermentation
 
One of the key technological properties of milk is its ability to form curds with desirable structural and mechanical characteristics under the action of rennet enzymes or enzymes produced by starter cultures. While this capacity has been extensively studied in cow’s milk-the conventional raw material in the dairy industry-donkey milk represents a promising subject for further exploration in this regard. The results obtained from such investigations can be regarded as both novel and of practical significance. Study confirmed the feasibility of using donkey milk as a suitable medium for the growth of starter microflora. A primary indicator of active lactobacilli proliferation-the main constituents of industrial starter cultures-is an increase in titratable acidity, driven by the accumulation of lactic acid produced during lactose fermentation. The rate of acidification and therefore the duration of the technological process, depends largely on the composition of the starter cultures.
       
We examined the acidifying potential of three different microbial combinations involving lactobacilli, bifidobacteria and propionic acid bacteria:
The first combination consisted of Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.
The second combination included the same microorganisms, with the addition of Bifidobacterium bifidum and Propionibacterium freudenreichii.
The third combination further expanded the microbiological consortium by adding Lactobacillus plantarum to the second combination.        
The specified combinations of starter microflora were introduced into pretreated donkey milk (designated as DM1, DM2 and DM3) as well as into the comparison control - cow’s milk (CM1, CM2 and CM3). The fermentation parameters for both milk types were standardized at a temperature of 35°C and a total duration of 8 hours.
       
Throughout the fermentation process, the primary monitored parameters were active acidity (Fig 1) and titratable acidity (Fig 2), both measured at one-hour intervals.

Fig 1: Dynamics of active acidity (pH) changes in cow milk (CM) and donkey milk (DM) during fermentation with different combinations of starter cultures (1, 2 and 3).



Fig 2: Dynamics of titratable acidity changes in cow milk (CM) and donkey milk (DM) during fermentation with different combinations of starter cultures (1, 2 and 3).


       
Analysis of the data presented in Fig 1 indicates that the pH of both cow and donkey milk steadily decreases during fermentation when inoculated with various combinations of lactobacilli and other probiotic microorganisms. A notable acceleration in acidification is observed between the third and fifth hours of incubation. This trend is likely attributed to the buffering capacity of milk proteins. Gel formation occurs at the isoelectric point -when the net charge on casein micelles reaches zero (pH 4.6-4.7) - that, across all tested samples, is typically achieved approximately six hours after inoculation.
       
In contrast, the dynamics of titratable acidity (Fig 2) exhibit a different pattern. The fermentation curves for different microbial groups clearly illustrate variations in fermentation rates and the accumulation of organic acids derived from lactose metabolism. These differences reflect the distinct biochemical behavior of cow and donkey milk under the influence of the respective microbial consortia.
       
According to GOST 33491-2015, the standardized range of titratable acidity for fermented milk beverages is 85-130°T. In this study, the target value of 120°T was achieved in cow’s milk after 6 hours of fermentation, regardless of the starter culture composition. In contrast, donkey milk samples inoculated with the first and second microbial combinations (DM1 and DM2) exhibited titratable acidity levels of 100°T and 80°T, respectively, at the same fermentation point-values that may enhance the dietary benefits of the final product. The inclusion of Lactobacillus plantarum in the third starter combination (DM3) resulted in an increased acidity of 130°T at hour six. This can be attributed to the high acidogenic potential of L. plantarum and its stimulatory effect on propionic acid bacteria, resulting in elevated acid concentrations. These findings align with the observations of Tarnaud et al., (2020), who demonstrated that co-cultivation of L. plantarum with propionic acid bacteria promotes their growth in both cow and soy milk likely due to the production of lactic acid, which creates a favorable environment for propionibacteria. Furthermore, it is documented that mixed cultures of Propionibacterium freudenreichii and L. plantarum are used in the baking industry as biopreservatives with antifungal properties (Ran et al., 2022), In addition, Propionibacterium freudenreichii has GRAS (generally recognized as safe) status (Tomar, 2024).
       
The slower rate of acidification observed in DM1 and DM2, despite donkey milk’s high lactose content, may be due to its inherent bactericidal properties, which potentially affect starter culture activity. Previous research has shown that mild heat treatment does not significantly reduce lysozyme activity in donkey milk (Wang et al., 2022).
       
By monitoring the shifts in active and titratable acidity during the fermentation of cow’s milk supplemented with various combinations of probiotic cultures, the coagulation intensity (Is) was calculated using Equation (1) (Fig 3) and the coagulation coefficient (δ) was determined using Equation (2) (Fig 4) (Topel, 2012; Elemanova et al., 2022). These parameters provide a more precise representation of the dynamics of lactic acid fermentation. Coagulation intensity is defined as the rate of lactic acid accumulation, expressed in °T per unit of time (Fig 3).

Fig 3: Fermentation intensity of cow milk (CM) and donkey milk (DM) by combined starters.



Fig 4: Fermentation coefficient of cow (CM) and donkey (DM) milk by combined starters.


       
The obtained data, reflecting the rate of lactic acid accumulation during the fermentation of cow and donkey milk (graphically presented in Fig 3), demonstrate the presence of three characteristic phases. For all samples, the coagulation intensity (Is) peaks and subsequently declines to near-zero levels. The first peak occurs at 60 minutes; the second appears between 240 minutes (in CM 2, DM 3, CM 3) and 300 minutes (in DM 1, CM 1, DM 2); the third is observed from 420 minutes (CM 1, CM 2, CM 3) to 480 minutes (DM 1).
       
The height of the peaks suggests that in the initial phase, the acidification process driven by the starter cultures proceeds more actively in cow’s milk compared to donkey milk. This is likely due to the pronounced bactericidal properties of donkey milk, specifically its high levels of lysozyme and lactoferrin (Papademas, 2021; Ashokkumar, 2011; Chandrashekar, 2018). Following a temporary decline in the rate of acid formation, a second period of intensified acid production begins, during which the Is values show variability in both timing and magnitude. Nevertheless, a peak in microbial activity is clearly observed between 240 and 300 minutes, with donkey milk reaching its maximum Is at the 300-minute mark. This is followed by a gradual decrease in the acidification rate, accompanied by a slight increase in the concentration of organic acids formed during fermentation.
       
As illustrated in Figure 4, the highest values of the coagulation coefficient (δ), calculated according to Equation (2), were observed in cow milk fermented with the second group of probiotic cultures and in donkey milk fermented with the third group. The presence of Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium and Lactobacillus plantarum-microorganisms known for their high lactose-to-lactic-acid conversion efficiency-may influence the metabolic and enzymatic activity of Propionibacterium.
 
Gel-forming during donkey milk fermentation
 
The biochemical processes of milk fermentation, accompanied by the formation of lactic and propionic acids, lead to gelation - an irreversible sol-to-gel transition. This process is driven by the cessation of carboxyl group dissociation in casein molecules as pH drops, reducing the net negative charge to zero at the isoelectric point (pH 4.6-4.7 for casein). The resulting decrease in electrostatic repulsion between casein micelles enhances their interaction, leading to the formation of a structured system -either a gel or a coagulated fermented matrix. In donkey milk, this process remains poorly understood, which provided the rationale for the present study aimed at identifying patterns of gel structure formation during fermentation of donkey milk using the tested starter culture combinations. To this end, during the biotransformation of milk samples induced by the three described starter culture combinations, the following rheological parameters were determined using an MCR 302 rheometer: Complex viscosity, storage modulus (G′) and loss modulus (G″).
       
The observed changes in complex viscosity during the fermentation of cow and donkey milk are shown in Fig 5.

Fig 5: Dynamics of viscosity increase during fermentation of milk samples with various combinations of starter cultures: (a) Cow milk, (b) Donkey milk.


       
Cow milk fermented with the third combination of cultures (CM 3) started to gel after 218 minutes of fermentation. In sample CM 2, which contained the same microorganisms except for Lactobacillus plantarum, the gelation process started 42 minutes later. In CM 1, which included Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, gelation occurred 10 minutes later than in CM 3 (Fig 5a). These findings align with the observed increase in acidity dynamics in the respective samples.
       
In donkey milk with the third culture combination (DM 3), a sharp rise in complex viscosity was observed as early as 138 minutes into fermentation, whereas in DM 1 and DM 2 this process began later-at 172 and 239 minutes, respectively (Fig 5b). It is suggested that the presence of Lactobacillus plantarum in the starter culture accelerates gel formation, consistent with the faster acidification observed in this sample (Fig 4). Notably, in donkey milk fermented with the third culture group, the increase in complex viscosity began 80 minutes earlier than in cow milk with the same microbial composition, despite the later onset of acidification.
       
The primary structural and mechanical characteristics of non-Newtonian viscoelastic systems, such as fermented milk beverages, are the storage modulus (G′) and the loss modulus (G″) (Elemanova et al., 2022; Smanalieva et al., 2021). These parameters were determined for donkey milk during fermentation using different groups of starter cultures: (a) First combination, (b) Second combination and (c) Third combination. The results are presented in Fig 7 and compared with corresponding data for cow milk (Fig 6).

Fig 6: Dynamics of change of storage modulus (G') and loss modulus (G'') during fermentation of cow milk samples with combined starters.



Fig 7: Dynamics of change of storage modulus (G') and loss modulus (G'') during fermentation of donkey milk samples with combined starters.


       
In the cow milk sample fermented with the third group of starter cultures (Fig 6c), the crossover point G′ = G″ occurred earlier than in the other samples-at 225 minutes. At the same time, the strength of the forming gel, assessed by the storage modulus (G′) at the end of fermentation, was the highest, reaching 72 Pa. During the fermentation of donkey milk with three different groups of starter cultures, the crossover point G′ = G″ occurred earlier at 150 minutes (Fig 7). However, the weak gel strength was recorded in the donkey milk sample fermented with the third group of cultures (2.8 Pa). Significantly lower values of complex viscosity and storage modulus for donkey milk gels compared to cow milk gels can be explained by the lower casein content in donkey milk, which is approximately 1.5%, whereas in cow milk, this component reaches 3.2%. The structural integrity of fermented donkey milk is notably lower than that of cow, yak, or khainak milks, which exhibit higher storage moduli and gel strength (84 Pa and 420 Pa, respectively), consistent with their distinct chemical constituents (Usubalieva et al., 2024; Elemanova et al., 2022). This contrast highlights the superior ability of cow and high-altitude milks to form a more robust coagulum compared to the donkey milk base.
       
Analysis of the obtained data on acidification and structure-formation processes in donkey milk under the influence of various combined starter cultures revealed that the optimal composition is the third combination, which includes Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium bifidum, Propionibacterium freudenreichii and Lactobacillus plantarum. This combination can be recommended for developing a new fermented dairy product based on donkey milk.
 
Gel-forming property of donkey milk supplemented with plant ingredients during fermentation
 
Plant ingredients introduced into the beverage can have positive, negative, or neutral effects on the activity of the starter microflora. To evaluate the influence of the plant component in the form of carrot puree on the fermentation process of donkey milk by the third combination of starter cultures, a study was conducted of the changes in rheological properties of donkey milk samples (DM 3) and the milk-vegetable mixture (DMV 3). Fig 8 shows changes in complex viscosity and Fig 9 shows changes in the loss and storage moduli during fermentation.

Fig 8: Comparison of changes in complex viscosity during fermentation of milk (DM 3) and milk-vegetable (DMV 3) mixtures with a combination of starter cultures.


       
Thus, the final complex viscosity of the donkey milk sample DM 3, equal to 0.45 Pa·s, at the end of fermentation, whereas the same viscosity value for sample DMV (milk-vegetable mixture) was observed much earlier, at the 270th minute (Fig 8). Furthermore, the final complex viscosity of the milk-vegetable mixture was 0.78 Pa·s, which is 1.7 times higher than that of the unsupplemented sample.
       
Fig 9 shows that the gel-forming onset for the donkey milk-vegetable mixture was characterized by a crossover point of G′ and G″ occurring at the 136th minute, indicating a faster structure-forming process compared to pure donkey milk, which is observed at 150th minutes. A substantial difference is also seen in the final values of the storage modulus: 2.8 Pa for gels from donkey milk and 4.8 Pa for gels from the milk-vegetable mixture. These findings are consistent with those of other researchers. According to Smanalieva et al. (2025), enrichment of milk with 1.5% starch-containing additives accelerated lactic acid formation and, therefore, casein coagulation occurred more quickly. This suggests that the enhanced gel strength is primarily due to carrot carbohydrates and starch acting as structural fillers and that their stimulating effect on the starter microflora accelerates gel forming.

Fig 9: Comparison of changes of storage modulus (G') and loss modulus (G'') during fermentation of milk (DM 3) and milk-vegetable (DMV 3) mixture with the combination of starter cultures.

CONCLUSION

The regularities of acid and structure formation during fermentation of donkey milk from the Kyrgyz population using combined starter cultures were investigated for the first time. It was established that the consortium exhibiting optimal acid-forming properties includes lactic acid and propionic acid bacteria: Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium bifidum, Propionibacterium freudenreichii and Lactobacillus plantarum. Rheological measurements in Time-Sweep mode, including determination of complex viscosity, storage modulus (G′) and loss modulus (G″), confirmed the effectiveness of this starter culture combination, enabling intensification of the fermented dairy beverage production process. The results provided a scientific basis for developing a new fermented beverage formulation and technology incorporating donkey milk and carrot puree. This approach not only enhances the product’s functional properties but also accelerates the technological process and improves the structural and textural characteristics of the resulting gels.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Funding
 
The study was carried out within the framework of the project funded by the Ministry of Education and Science of the Kyrgyz Republic (grant number 007652).

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest regarding the publication of this research paper.

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    Full Research Article

    Fermentation-induced Acidification and Structure Formation in Donkey Milk

    N
    N.K. Turganbaeva1,*
    0000-0002-7620-9236
    R
    R. Sh. Elemanova2
    J
    J.N. Smanalieva2,3
    M
    M.M. Musulmanova2
    1Kyrgyz-Turkish Manas University, Bishkek, Kyrgyz Republic.
    2I. Razzakov Kyrgyz State Technical University, Bishkek, Kyrgyz Republic.
    3Technical University Dresden, Dresden, Germany.

    ABSTRACT

    Background: The search for new physiologically functional ingredients has led to increased attention toward donkey milk, recognized as a promising raw material for various food products, including fermented ones. This study investigates the potential of donkey milk for fermented dairy production by examining curd formation during milk biotransformation with different starter cultures.

    Methods: The study to evaluate acid-forming properties of donkey milk employed the following starter cultures: Lactobacillus acidophilus, Bifidobacterium subsp. lactis, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Additionally, bacterial concentrates of Lactobacillus plantarum and Propionibacterium freudenreichii were used. Rheological parameters were determined using oscillatory measurement.

    Result: Industrial starter cultures exhibited high growth and biochemical activity in donkey milk. A consortium of lactic and propionic acid bacteria achieved optimal acidification and coagulation. For the first time, rheological analysis - including complex viscosity, storage and loss moduli - characterized donkey milk curd, confirming its suitability for fermented products. Results showed that specific culture combinations effectively regulate titratable acidity and curdling coefficients. These findings established the technological basis for a novel fermented milk-plant beverage, demonstrating the potential of donkey milk as a functional dairy base.

    INTRODUCTION

    In recent years, consumer interest in donkey milk has steadily increased and it is gaining popularity across various markets. This product is notable not only for its distinctive taste but also for its high content of biologically valuable components. Despite its favorable nutritional profile, donkey milk was previously considered a niche product with limited accessibility for the public. However, the growth of e-commerce has significantly increased its availability, as many producers now market donkey milk through online platforms that offer international delivery. The growing consumer loyalty toward donkey milk reflects a broader global trend toward the consumption of natural and functional food products. In the Kyrgyz Republic, there is also a noticeable rise in interest in donkey milk as a source of bioactive compounds. Although religious dietary restrictions classify donkey milk as haram, its consumption for wellness purposes is increasing, often accompanied by positive anecdotal reports of its health benefits.
           
    It is well established that human, donkey and mare’s milk are characterized by high biological and nutritional value, owing to a well-balanced amino acid composition and elevated lactose content (Felicita et al., 2014, Madhusudan et al., 2020; Altomonte et al., 2019; Giosue et al., 2008; Wszołek et al., 2014; Bhardwaj et al., 2019). Furthermore, these types of milk are rich in α-albumin, with an average particle size not exceeding 15-20 nm (Zhang et al., 2020; Ostroumova, 2004). The proportion of whey proteins in donkey and mare’s milk accounts for 35-50% of total protein content (Felicita et al., 2014; Madhusudan et al., 2020; Giosue et al., 2008; Wszołek et al., 2014), which categorizes them, along with human milk, as albuminous types. The low casein content promotes the formation of fine, soft curds during gastric coagulation, which are easily digestible and suitable for infant nutrition (Gorbatova, 2010). Moreover, the significantly reduced casein concentration in donkey milk contributes to its hypoallergenic properties, making it a valuable alternative for individuals with cow’s milk protein allergy (Cunsolo et al., 2017; Vincenzetti et al., 2014; Polidori et al., 2015; Prasad, 2020; Nayak, 2020).
           
    The high levels of lactose and lysozyme in donkey milk open up opportunities for the development of new types of fermented dairy products (Cavalcanti et al., 2021; Vincenzetti et al., 2017; Dambrosio et al., 2023; Perna et al., 2015). Fermented dairy products are considered an optimal dietary option for individuals with lactose intolerance, as the fermentation process breaks down lactose into lactic acid, which positively influences the secretory function of the gastrointestinal tract. Lactose is the main substrate for acid production during fermentation, converted into lactic acid by the enzymatic activity of lactic acid bacteria in starter cultures.
           
    The use of probiotic starter cultures is driven by their well-documented health benefits, including immunomodulatory, antimicrobial and antioxidant properties (Cousin et al., 2012; Hashemi et al., 2023; Rosa et al., 2022). The potential of Streptococcus thermophilus and Bifidobacterium subsp. Lactis strains involved in folate biosynthesis were investigated and it was found that when they are cultivated together, folate concentrations in the final product can increase sixfold (Adriani, 2024). These microorganisms play a crucial role in enhancing the nutritional value of fermented products and supporting gastrointestinal health (Yang et al., 2019; Mantel et al., 2022). Starter cultures play a crucial role in shaping the organoleptic characteristics of fermented dairy products. Lactic acid, produced during fermentation, functions as a natural preservative, enhancing the freshness and microbiological safety of the final product. However, the behavior of lactic acid bacteria and other microorganisms in starter cultures when introduced into donkey milk from the Kyrgyz population remains largely unexplored. This formed the basis of our study. The findings provide valuable insight into the technological properties of donkey milk as a substrate that supports the growth of starter microflora and the associated accumulation of lactic acid, leading to casein coagulation and the formation of a fermented milk curd.

    MATERIALS AND METHODS

    The work was carried out in the Department of Food Production Technology at the Technological Institute of the Kyrgyz State Technical University named after I. Razzakov during 2022-2023.
           
    Donkey milk samples were obtained from two clinically healthy, lactating Kyrgyz donkeys aged 5 years, kept on a farm belonging to the Karà-Zhygach state agricultural enterprise in the Chüy Valley of the Kyrgyz Republic, at an altitude of 800 meters above sea level.
           
    The preparation of milk samples for fermentation involved mild pasteurization (75°C for 15 minutes) (Luo et al., 2019, Giacometti et al., 2016; Wang et al., 2022), which was selected due to the low thermal stability of this type of milk. Following heat treatment, the samples were cooled to the inoculation temperature of 35°C.
           
    The physicochemical characteristics of donkey milk from Kyrgyzstan are presented in Table 1.

    Table 1: Physical and chemical composition of donkey milk from Kyrgyzstan.


           
    As a comparative control, ultra-pasteurized cow’s milk of the brand “Vesyolyy Molochnik” was used produced by “Bishkeksut”, with a fat content of 1.5%, protein 2.9% and carbohydrates 4.7%.
           
    To investigate the acid-forming properties of the starter cultures, the following microorganisms were utilized: Lactobacillus acidophilus (probiotic), Bifidobacterium subsp. Lactis (probiotic), Streptococcus thermophilus (probiotic) “Lyofast SAB 439 A” and Lactobacillus delbrueckii subsp. bulgaricus (probiotic) “Lyofast SAB 438B” (BioChem, Italy), each containing not fewer than 1011 CFU/g. In addition, a bacterial concentrate of Lactobacillus plantarum (probiotic) with a cell concentration of at least 1010 CFU/g, including phage-resistant strains for prolonged use (BioChem, Italy), was employed. Also included was a Propionibacterium freudenreichii (probiotic) bacterial concentrate (BioChem, Italy) with a cell density of at least 3×1010 CFU/g. The amount of inoculated culture followed the manufacturer’s recommended dosage.
           
    The optimal growth temperature range for these microorganisms was between 30 and 45°C.

    The rate of acidification, expressed in °T per unit of time, was determined using Equation (1) (Topel, 2012; Elemanova et al., 2022).
     
                 Is = TAC2 - TAC1                          ...(1)
     
    Where,
    TAC1- Titratable acidity at the beginning of the observation period (°T).
    TAC2- Titratable acidity at the end of the observation period (°T).
           
    The acidification coefficient, which reflects the intensity of acidification relative to the observed increase in titratable acidity, was calculated using Equation (2) (Topel, 2012; Elemanova et al., 2022).

     
    Where,
    ΔTAC2- The difference between the initial acidity and the acidity measured at the end of the observation period (°T).
    ΔTAC1- The difference between the initial acidity and the acidity measured at the beginning of the observation period (°T).
     
    Rheological measurements
     
    Rheological properties, namely complex viscosity (η), storage modulus (G′) and loss modulus (G″), were assessed by oscillatory measurement on the MCR 302 rheometer (Anton Paar, RheoCompass, Austria) using a time-sweep test, following the procedure described by Smanalieva et al. (2021). Data acquisition and processing were carried out with RheoCompass software, version 1.30. Milk samples were inoculated with the combined starter cultures and, at 35°C, transferred to the rheometer’s measuring cup. Rheological measurements were conducted over an 8-hour period under the following oscillation settings: strain amplitude γ = 1 %, frequency f = 1 Hz, employing a concentric-cylinder geometry (CC27-SN26341).
           
    The formulation of the functional beverage was optimized using a linear programming mathematical model via LINDO software (Lindo Systems, 2020). Based on the optimization results, the optimal ratio of donkey milk to carrot puree was determined to be 70:30 (v/v).
           
    Statistical analysis was performed in SPSS 22 (SPSS Inc., Chicago, IL) using paired-sample t-tests with a 95% confidence interval; differences were considered statistically significant at p<0.05.

    RESULTS AND DISCUSSION

    Acid formation during donkey milk fermentation
     
    One of the key technological properties of milk is its ability to form curds with desirable structural and mechanical characteristics under the action of rennet enzymes or enzymes produced by starter cultures. While this capacity has been extensively studied in cow’s milk-the conventional raw material in the dairy industry-donkey milk represents a promising subject for further exploration in this regard. The results obtained from such investigations can be regarded as both novel and of practical significance. Study confirmed the feasibility of using donkey milk as a suitable medium for the growth of starter microflora. A primary indicator of active lactobacilli proliferation-the main constituents of industrial starter cultures-is an increase in titratable acidity, driven by the accumulation of lactic acid produced during lactose fermentation. The rate of acidification and therefore the duration of the technological process, depends largely on the composition of the starter cultures.
           
    We examined the acidifying potential of three different microbial combinations involving lactobacilli, bifidobacteria and propionic acid bacteria:
    The first combination consisted of Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.
    The second combination included the same microorganisms, with the addition of Bifidobacterium bifidum and Propionibacterium freudenreichii.
    The third combination further expanded the microbiological consortium by adding Lactobacillus plantarum to the second combination.        
    The specified combinations of starter microflora were introduced into pretreated donkey milk (designated as DM1, DM2 and DM3) as well as into the comparison control - cow’s milk (CM1, CM2 and CM3). The fermentation parameters for both milk types were standardized at a temperature of 35°C and a total duration of 8 hours.
           
    Throughout the fermentation process, the primary monitored parameters were active acidity (Fig 1) and titratable acidity (Fig 2), both measured at one-hour intervals.

    Fig 1: Dynamics of active acidity (pH) changes in cow milk (CM) and donkey milk (DM) during fermentation with different combinations of starter cultures (1, 2 and 3).



    Fig 2: Dynamics of titratable acidity changes in cow milk (CM) and donkey milk (DM) during fermentation with different combinations of starter cultures (1, 2 and 3).


           
    Analysis of the data presented in Fig 1 indicates that the pH of both cow and donkey milk steadily decreases during fermentation when inoculated with various combinations of lactobacilli and other probiotic microorganisms. A notable acceleration in acidification is observed between the third and fifth hours of incubation. This trend is likely attributed to the buffering capacity of milk proteins. Gel formation occurs at the isoelectric point -when the net charge on casein micelles reaches zero (pH 4.6-4.7) - that, across all tested samples, is typically achieved approximately six hours after inoculation.
           
    In contrast, the dynamics of titratable acidity (Fig 2) exhibit a different pattern. The fermentation curves for different microbial groups clearly illustrate variations in fermentation rates and the accumulation of organic acids derived from lactose metabolism. These differences reflect the distinct biochemical behavior of cow and donkey milk under the influence of the respective microbial consortia.
           
    According to GOST 33491-2015, the standardized range of titratable acidity for fermented milk beverages is 85-130°T. In this study, the target value of 120°T was achieved in cow’s milk after 6 hours of fermentation, regardless of the starter culture composition. In contrast, donkey milk samples inoculated with the first and second microbial combinations (DM1 and DM2) exhibited titratable acidity levels of 100°T and 80°T, respectively, at the same fermentation point-values that may enhance the dietary benefits of the final product. The inclusion of Lactobacillus plantarum in the third starter combination (DM3) resulted in an increased acidity of 130°T at hour six. This can be attributed to the high acidogenic potential of L. plantarum and its stimulatory effect on propionic acid bacteria, resulting in elevated acid concentrations. These findings align with the observations of Tarnaud et al., (2020), who demonstrated that co-cultivation of L. plantarum with propionic acid bacteria promotes their growth in both cow and soy milk likely due to the production of lactic acid, which creates a favorable environment for propionibacteria. Furthermore, it is documented that mixed cultures of Propionibacterium freudenreichii and L. plantarum are used in the baking industry as biopreservatives with antifungal properties (Ran et al., 2022), In addition, Propionibacterium freudenreichii has GRAS (generally recognized as safe) status (Tomar, 2024).
           
    The slower rate of acidification observed in DM1 and DM2, despite donkey milk’s high lactose content, may be due to its inherent bactericidal properties, which potentially affect starter culture activity. Previous research has shown that mild heat treatment does not significantly reduce lysozyme activity in donkey milk (Wang et al., 2022).
           
    By monitoring the shifts in active and titratable acidity during the fermentation of cow’s milk supplemented with various combinations of probiotic cultures, the coagulation intensity (Is) was calculated using Equation (1) (Fig 3) and the coagulation coefficient (δ) was determined using Equation (2) (Fig 4) (Topel, 2012; Elemanova et al., 2022). These parameters provide a more precise representation of the dynamics of lactic acid fermentation. Coagulation intensity is defined as the rate of lactic acid accumulation, expressed in °T per unit of time (Fig 3).

    Fig 3: Fermentation intensity of cow milk (CM) and donkey milk (DM) by combined starters.



    Fig 4: Fermentation coefficient of cow (CM) and donkey (DM) milk by combined starters.


           
    The obtained data, reflecting the rate of lactic acid accumulation during the fermentation of cow and donkey milk (graphically presented in Fig 3), demonstrate the presence of three characteristic phases. For all samples, the coagulation intensity (Is) peaks and subsequently declines to near-zero levels. The first peak occurs at 60 minutes; the second appears between 240 minutes (in CM 2, DM 3, CM 3) and 300 minutes (in DM 1, CM 1, DM 2); the third is observed from 420 minutes (CM 1, CM 2, CM 3) to 480 minutes (DM 1).
           
    The height of the peaks suggests that in the initial phase, the acidification process driven by the starter cultures proceeds more actively in cow’s milk compared to donkey milk. This is likely due to the pronounced bactericidal properties of donkey milk, specifically its high levels of lysozyme and lactoferrin (Papademas, 2021; Ashokkumar, 2011; Chandrashekar, 2018). Following a temporary decline in the rate of acid formation, a second period of intensified acid production begins, during which the Is values show variability in both timing and magnitude. Nevertheless, a peak in microbial activity is clearly observed between 240 and 300 minutes, with donkey milk reaching its maximum Is at the 300-minute mark. This is followed by a gradual decrease in the acidification rate, accompanied by a slight increase in the concentration of organic acids formed during fermentation.
           
    As illustrated in Figure 4, the highest values of the coagulation coefficient (δ), calculated according to Equation (2), were observed in cow milk fermented with the second group of probiotic cultures and in donkey milk fermented with the third group. The presence of Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium and Lactobacillus plantarum-microorganisms known for their high lactose-to-lactic-acid conversion efficiency-may influence the metabolic and enzymatic activity of Propionibacterium.
     
    Gel-forming during donkey milk fermentation
     
    The biochemical processes of milk fermentation, accompanied by the formation of lactic and propionic acids, lead to gelation - an irreversible sol-to-gel transition. This process is driven by the cessation of carboxyl group dissociation in casein molecules as pH drops, reducing the net negative charge to zero at the isoelectric point (pH 4.6-4.7 for casein). The resulting decrease in electrostatic repulsion between casein micelles enhances their interaction, leading to the formation of a structured system -either a gel or a coagulated fermented matrix. In donkey milk, this process remains poorly understood, which provided the rationale for the present study aimed at identifying patterns of gel structure formation during fermentation of donkey milk using the tested starter culture combinations. To this end, during the biotransformation of milk samples induced by the three described starter culture combinations, the following rheological parameters were determined using an MCR 302 rheometer: Complex viscosity, storage modulus (G′) and loss modulus (G″).
           
    The observed changes in complex viscosity during the fermentation of cow and donkey milk are shown in Fig 5.

    Fig 5: Dynamics of viscosity increase during fermentation of milk samples with various combinations of starter cultures: (a) Cow milk, (b) Donkey milk.


           
    Cow milk fermented with the third combination of cultures (CM 3) started to gel after 218 minutes of fermentation. In sample CM 2, which contained the same microorganisms except for Lactobacillus plantarum, the gelation process started 42 minutes later. In CM 1, which included Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, gelation occurred 10 minutes later than in CM 3 (Fig 5a). These findings align with the observed increase in acidity dynamics in the respective samples.
           
    In donkey milk with the third culture combination (DM 3), a sharp rise in complex viscosity was observed as early as 138 minutes into fermentation, whereas in DM 1 and DM 2 this process began later-at 172 and 239 minutes, respectively (Fig 5b). It is suggested that the presence of Lactobacillus plantarum in the starter culture accelerates gel formation, consistent with the faster acidification observed in this sample (Fig 4). Notably, in donkey milk fermented with the third culture group, the increase in complex viscosity began 80 minutes earlier than in cow milk with the same microbial composition, despite the later onset of acidification.
           
    The primary structural and mechanical characteristics of non-Newtonian viscoelastic systems, such as fermented milk beverages, are the storage modulus (G′) and the loss modulus (G″) (Elemanova et al., 2022; Smanalieva et al., 2021). These parameters were determined for donkey milk during fermentation using different groups of starter cultures: (a) First combination, (b) Second combination and (c) Third combination. The results are presented in Fig 7 and compared with corresponding data for cow milk (Fig 6).

    Fig 6: Dynamics of change of storage modulus (G') and loss modulus (G'') during fermentation of cow milk samples with combined starters.



    Fig 7: Dynamics of change of storage modulus (G') and loss modulus (G'') during fermentation of donkey milk samples with combined starters.


           
    In the cow milk sample fermented with the third group of starter cultures (Fig 6c), the crossover point G′ = G″ occurred earlier than in the other samples-at 225 minutes. At the same time, the strength of the forming gel, assessed by the storage modulus (G′) at the end of fermentation, was the highest, reaching 72 Pa. During the fermentation of donkey milk with three different groups of starter cultures, the crossover point G′ = G″ occurred earlier at 150 minutes (Fig 7). However, the weak gel strength was recorded in the donkey milk sample fermented with the third group of cultures (2.8 Pa). Significantly lower values of complex viscosity and storage modulus for donkey milk gels compared to cow milk gels can be explained by the lower casein content in donkey milk, which is approximately 1.5%, whereas in cow milk, this component reaches 3.2%. The structural integrity of fermented donkey milk is notably lower than that of cow, yak, or khainak milks, which exhibit higher storage moduli and gel strength (84 Pa and 420 Pa, respectively), consistent with their distinct chemical constituents (Usubalieva et al., 2024; Elemanova et al., 2022). This contrast highlights the superior ability of cow and high-altitude milks to form a more robust coagulum compared to the donkey milk base.
           
    Analysis of the obtained data on acidification and structure-formation processes in donkey milk under the influence of various combined starter cultures revealed that the optimal composition is the third combination, which includes Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium bifidum, Propionibacterium freudenreichii and Lactobacillus plantarum. This combination can be recommended for developing a new fermented dairy product based on donkey milk.
     
    Gel-forming property of donkey milk supplemented with plant ingredients during fermentation
     
    Plant ingredients introduced into the beverage can have positive, negative, or neutral effects on the activity of the starter microflora. To evaluate the influence of the plant component in the form of carrot puree on the fermentation process of donkey milk by the third combination of starter cultures, a study was conducted of the changes in rheological properties of donkey milk samples (DM 3) and the milk-vegetable mixture (DMV 3). Fig 8 shows changes in complex viscosity and Fig 9 shows changes in the loss and storage moduli during fermentation.

    Fig 8: Comparison of changes in complex viscosity during fermentation of milk (DM 3) and milk-vegetable (DMV 3) mixtures with a combination of starter cultures.


           
    Thus, the final complex viscosity of the donkey milk sample DM 3, equal to 0.45 Pa·s, at the end of fermentation, whereas the same viscosity value for sample DMV (milk-vegetable mixture) was observed much earlier, at the 270th minute (Fig 8). Furthermore, the final complex viscosity of the milk-vegetable mixture was 0.78 Pa·s, which is 1.7 times higher than that of the unsupplemented sample.
           
    Fig 9 shows that the gel-forming onset for the donkey milk-vegetable mixture was characterized by a crossover point of G′ and G″ occurring at the 136th minute, indicating a faster structure-forming process compared to pure donkey milk, which is observed at 150th minutes. A substantial difference is also seen in the final values of the storage modulus: 2.8 Pa for gels from donkey milk and 4.8 Pa for gels from the milk-vegetable mixture. These findings are consistent with those of other researchers. According to Smanalieva et al. (2025), enrichment of milk with 1.5% starch-containing additives accelerated lactic acid formation and, therefore, casein coagulation occurred more quickly. This suggests that the enhanced gel strength is primarily due to carrot carbohydrates and starch acting as structural fillers and that their stimulating effect on the starter microflora accelerates gel forming.

    Fig 9: Comparison of changes of storage modulus (G') and loss modulus (G'') during fermentation of milk (DM 3) and milk-vegetable (DMV 3) mixture with the combination of starter cultures.

    CONCLUSION

    The regularities of acid and structure formation during fermentation of donkey milk from the Kyrgyz population using combined starter cultures were investigated for the first time. It was established that the consortium exhibiting optimal acid-forming properties includes lactic acid and propionic acid bacteria: Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium bifidum, Propionibacterium freudenreichii and Lactobacillus plantarum. Rheological measurements in Time-Sweep mode, including determination of complex viscosity, storage modulus (G′) and loss modulus (G″), confirmed the effectiveness of this starter culture combination, enabling intensification of the fermented dairy beverage production process. The results provided a scientific basis for developing a new fermented beverage formulation and technology incorporating donkey milk and carrot puree. This approach not only enhances the product’s functional properties but also accelerates the technological process and improves the structural and textural characteristics of the resulting gels.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Funding
     
    The study was carried out within the framework of the project funded by the Ministry of Education and Science of the Kyrgyz Republic (grant number 007652).

    CONFLICT OF INTEREST

    The authors declare that there is no conflict of interest regarding the publication of this research paper.

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