Storage stability and quality characteristics of oat powder incorporated probiotic Shrikhand prepared using multi-strain probiotic culture

Abstract

This study investigated the storage stability, physicochemical properties, probiotic viability, microbiological, and sensory quality of oat powder incorporated probiotic Shrikhand prepared using ABY-3 multi-strain culture. Fresh cow milk was fermented using ABY-3 culture, chakka was prepared, and Shrikhand was formulated with 2% oat powder and 40% sugar. A second inoculation step ensured initial probiotic counts of 10⁸–10⁹ CFU/g. Samples were stored at 4 ± 1 °C and analyzed on days 0, 7, 14, 21, 28, and 35. Post-acidification was evident through a significant pH decline (4.37 to 4.06) and an increase in titratable acidity (0.98 to 1.33% LA) over 35 days. Oat β-glucans contributed to a slight but consistent reduction in water activity (0.9391 to 0.9330) and enhanced structural stability, reflected in steadily increasing firmness values (7.81 to 9.23 N). Free fatty acids (0.64 to 1.13 µ eq. KOH/g) and soluble nitrogen (0.24 to 0.44%) increased progressively. Probiotic counts declined gradually (8.73 to 7.50 log CFU/g) yet remained above the functional threshold (> 10⁶–10⁷ CFU/g) throughout storage. Standard Plate Count increased moderately but remained within acceptable limits. Yeast and mould growth appeared after day 7 but did not reach spoilage levels. Sensory scores decreased significantly with storage, with overall acceptability remaining above 80 until day 21, after which rising acidity and firmer texture caused noticeable quality deterioration. Oat powder fortification notably enhanced water-binding capacity, structural stability, and microbial quality of probiotic Shrikhand without affecting sensory characteristics. The product retained functional probiotic levels, acceptable physicochemical properties, and desirable sensory attributes for up to 21–28 days of refrigerated storage, demonstrating its suitability as a clean-label, fiber-enriched functional dairy dessert. The findings provide valuable insights for commercial development of oat powder incorporated probiotic Shrikhand with improved shelf-life and nutritional functionality.

Introduction

Shrikhand is a traditional semi-solid fermented dairy product widely consumed in Western India, particularly in Gujarat and Maharashtra, where it is valued for its rich flavor and smooth texture. Shrikhand is produced by partial removal of whey from fermented milk (curd) to obtain chakka and blending with sugar^1–3. As consumers increasingly prioritize nutritional functionality, gastrointestinal health, and clean-label ingredients, Shrikhand has gained attention as a platform for incorporating dietary fiber and probiotic cultures, thereby transforming a traditional delicacy into a scientifically designed functional food⁴.

The incorporation of dietary fiber into dairy-based fermented foods has emerged as an important approach to enhance their nutritional value, improve physiological responses, and modify functional attributes, largely because dairy products are widely consumed, economically accessible, and possess intrinsic structural and compositional characteristics that facilitate the incorporation, stability, and digestibility of added fiber⁴. Dietary fibers, especially soluble fibers, influence gastrointestinal function, attenuate glycemic spikes, and support the growth of beneficial microorganisms⁴. Among various fibers, oat-based fibers rich in β-glucans have been extensively researched due to their cholesterol-lowering properties, immunomodulatory effects, and viscosity-enhancing behavior^5–7. Oat β-glucans are linear polysaccharides containing β-(1→3) and β-(1→4) linkages that allow them to form highly hydrated, gel-like networks capable of retaining substantial amounts of water. This property is particularly valuable in fermented dairy systems that are susceptible to syneresis, as β-glucans interact with water molecules and the protein matrix to improve structural stability^7–11. Hence, incorporating oat powder into Shrikhand can therefore provide a multifunctional role. However, despite the rising interest, the scientific literature on the storage behavior of oat powder incorporated Shrikhand remains limited, with most available studies focusing only on conventional Shrikhand or those incorporated with inulin, guar gum, or psyllium⁴. The properties of oat β-glucans differ considerably from such fibers, particularly in terms of molecular weight distribution, solubility, rheology, and interactions with dairy proteins during storage^7–11.

Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host.”^12,13 Dairy products are the most widely accepted carriers for probiotics due to their nutrient density, near-neutral pH, lactose content, and buffering capacity, which collectively create a conducive environment for the growth and survival of probiotic strains^14,15. The ABY-3 culture system contains a synergistic combination of probiotic organisms including Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Bifidobacterium spp. This culture blend is extensively used in dairy fermentation due to its balanced metabolic interactions¹⁶. S. thermophilus and L. delbrueckii subsp. bulgaricus initiate fermentation, generating exopolysaccharides (EPS) that improve texture and minimize whey separation, while L. acidophilus and Bifidobacterium spp. contribute targeted health benefits related to immune regulation, intestinal barrier enhancement, and reduction of pathogenic bacteria¹⁷. In Shrikhand, combining oat powder with probiotics may create synergistic interaction because β-glucans act as prebiotic substrates that selectively enhance the activity and survival of probiotics^9,10. However, incorporating probiotics into Shrikhand introduces challenges such as post-acidification, which affects flavor, firmness, and sensory characteristics during storage^18,19.

Storage stability of probiotic dairy products is governed by physicochemical and microbiological changes during refrigerated storage (4 °C). Residual metabolic activity of Lactobacillus and Streptococcus causes post-acidification, leading to lactic acid accumulation, reduced pH, and release of bound water from casein micelles, which results in textural changes and reduced sensory acceptability^17,19. Oat β-glucan rich powder can mitigate such effects by binding water, limiting substrate diffusion, and restricting microbial mobility^9,10. Moreover, the incorporation of oat powder enhances water retention through hydrogen bonding, increased viscosity, and formation of β-glucan–casein networks that entrap the serum phase, thereby reducing syneresis and improving shelf-life stability in Shrikhand^1,8–10.

Texture, a critical sensory attribute determining consumer preference, also undergoes transformation during storage due to continued protein hydration, acid-induced protein aggregation, and rearrangement of casein–milk fat–sugar networks^20–22. Oat β-glucans contribute additional viscosity, potentially stabilizing the structure and minimizing texture-related defects. Interactions between oat fiber and milk proteins, especially β-casein and κ-casein, can enhance microstructural stability through hydrogen bonding interactions, steric entanglement, and co-network formation^8–11. Furthermore, several factors are affecting the survivability of probiotic in Shrikhand which include pH decline, exposure to oxygen, storage temperature fluctuations, and competition among co-cultured organisms¹³. Oat β-glucans may improve viability by acting as a carbon source for select microbes and reducing oxygen diffusion in the matrix due to enhanced viscosity^8–11. Different strains within ABY-3 exhibit varied survival characteristics; Bifidobacterium spp. are particularly sensitive to oxygen and acidic environments, while Streptococcus thermophilus generally shows good survival, emphasizing the need to consider strain-specific viability trends^16,17.

The sensory quality of Shrikhand depends on its sweetness, acidity, flavor, color, and texture or mouthfeel^1,2. Oat β-glucans, if not properly hydrated or uniformly dispersed, may impart a gritty sensation. Therefore, balanced formulation is necessary to ensure desirable functional and sensory attributes. Despite increasing consumer interest, systematic research on oat powder–incorporated probiotic Shrikhand remains scarce. The key knowledge gaps include understanding interaction mechanisms between oat fiber components and the fermented milk protein matrix, determining the impact on probiotic survival especially for mixed-strain cultures such as ABY-3, describing consistency during storage and determining shelf-life based on sensory, microbiological, and physical-chemical properties.

Therefore, the present study was designed to evaluate the storage stability of oat powder incorporated probiotic Shrikhand using the ABY-3 culture system. The specific objectives were to assess the physicochemical changes during 28 days of refrigerated storage; determine the viability of probiotic strains during storage; assess firmness, and sensory properties to determine consumer acceptability; and identify the mechanisms underlying storage-induced transitions, including acidification effects, and fiber–protein interactions.

Materials and methods

Materials

Fresh cow milk was procured from a local ISO-certified dairy plant and standardized to 4.5% fat and 8.5% solids-not-fat (SNF) prior to experimentation. All raw cow milk was screened for acidity, clot-on-boiling (COB), and microbial quality to ensure compliance with technological requirements for fermented milk production. Food-grade oat powder, characterized by a high soluble fiber fraction and enriched in β-glucans (≥ 4–6% w/w), was sourced from a certified commercial supplier (MRK Agrotech LLP, Anand, Gujarat, India). The oat powder was stored under cool, dry conditions (≤ 20 °C) and sieved (60 mesh) prior to use to ensure uniform particle distribution and prevent lump formation in the Shrikhand. A commercial ABY-3 multi-strain probiotic culture (Chr. Hansen Ltd., Mumbai, Maharastra, India) was used as the inoculum. The culture was stored at − 18 °C until use and added at manufacturer-recommended dosage to achieve an initial viable count of 10⁸–10⁹ CFU/g in the final product. Commercial-grade crystalline sugar (sucrose) and natural cardamom (Elettaria cardamomum) flavor were used as sweetening and flavoring agents. All additional chemicals and reagents employed in physicochemical and microbiological analyses, such as sodium hydroxide, phenolphthalein, buffer salts, agar media, and solvents, were of analytical grade and procured from reputed suppliers (HiMedia Laboratories, Mumbai, Maharastra, India). Sterile high-density polyethylene (HDPE) cups with tight-fitting lids were used for packaging Shrikhand samples during storage studies.

Preparation of oat-powder incorporated probiotic Shrikhand

Fresh cow milk was standardized to 4.5% fat and 8.5% SNF and subjected to thermal treatment at 90 °C for 10 min to pasteurize milk, denature whey proteins, enhance water-binding capacity, and improve the textural stability of the final product. Heat-treated milk was rapidly cooled to 42 ± 1 °C, the optimal temperature for mesophilic–thermophilic synergistic growth of the ABY-3 cultures. The milk was inoculated with ABY-3 DVS culture at 2% (w/v) and incubated undisturbed in a thermostatically controlled incubator until the curd achieved a firm, uniform coagulum with pH 4.4–4.5, indicating completion of fermentation. Post-incubation, the coagulated milk (dahi) was refrigerated at 7 °C for 1 h to strengthen the curd matrix and facilitate whey expulsion. The curd was transferred to a muslin cloth and allowed to drain by gravity for 12 h at 7–10 °C, yielding chakka. Shrikhand was prepared by blending chakka and granulated sugar at a 60:40 ratio (w/w) using a mechanical beater (1,200 rpm) for 4–5 min until a smooth, homogenous consistency was obtained. Food-grade oat powder (2% w/w) was gradually incorporated during blending to prevent lump formation. The addition of oat powder increased the soluble fiber content and contributed to viscosity, water-binding, and structural reinforcement. A second inoculation step was applied by adding 0.5% (v/w) of reactivated ABY-3 culture to the Shrikhand base to ensure high probiotic viability (> 10⁸ CFU/g) in the final product. Natural cardamom flavor (0.03% v/w) was incorporated to enhance sensory attributes without interfering with fermentation or storage behavior. The final product was immediately packaged into sterile, airtight HDPE cups (100 g) using a laminar flow workstation to minimize microbial contamination. Packaged samples were stored at 4 ± 1 °C and subjected to quality assessments at pre-determined intervals⁴.

Storage study design

Storage stability study was conducted to evaluate the changes in quality attributes of the oat powder (2%) incorporated probiotic Shrikhand under controlled refrigeration. All samples were stored at 4 ± 1 °C, simulating commercial cold-chain conditions typically maintained throughout distribution and consumer handling. Sampling was performed at five predetermined intervals, day 0 (freshly prepared), 7, 14, 21, and 28. These intervals were selected to capture the early, intermediate, and late phases of product deterioration, metabolic activity, and structural transitions occurring during refrigerated storage.

Physicochemical analyses

pH

pH was measured using a calibrated digital pH meter (Eutech Instruments, Singapore) with automatic temperature correction. Before each measurement, the electrode was calibrated using standard buffer solutions (pH 4.0 and pH 7.0). 10 g of sample was homogenized, and the electrode was inserted directly into the sample to avoid aeration and temperature alternations.

Titratable acidity

TA was determined by titration with 0.1 N NaOH, employing phenolphthalein as an indicator. About 10 g of Shrikhand was diluted (1:10) with distilled water and titrated until the first persistent faint pink color appeared. Results were expressed as % lactic acid, reflecting the total acid content and microbial metabolic activity during storage⁴.

Free fatty acids

Free fatty acids in Shrikhand were quantified using the solvent extraction and titrimetric procedure described by Verma et al.²³, with minor modifications due to the semi-solid nature of the product. A 3 g aliquot of Shrikhand was weighed into a clean, dry, 60 mL screw-cap centrifuge tube, after which 10 mL of extraction mixture (isopropanol: petroleum ether: 4 N H₂SO₄ in a ratio of 40:10:1 v/v/v), 6 mL of petroleum ether, and 4 mL of distilled water were added. The tube was securely stoppered and vigorously shaken for 15 s to ensure efficient extraction of liberated fatty acids. The mixture was then allowed to stand undisturbed for 5–10 min to achieve complete phase separation. A 7.5 mL aliquot of the upper organic layer was carefully transferred into a 50 mL Erlenmeyer flask using a glass pipette, avoiding contamination from the aqueous phase. To this aliquot, 6 drops of 1% methanolic phenolphthalein were added as an indicator. The extracted fatty acids were titrated against standardized 0.02 N methanolic KOH solution until the appearance of a stable pale-pink endpoint, persisting for at least 15 s.

Soluble nitrogen

Soluble nitrogen in Shrikhand samples was determined following the procedure described by Suvera et al.⁴, with slight modifications to suit the semi-solid nature of the product. 3 g of sample was accurately weighed into a clean beaker and mixed thoroughly with 40 mL of Sharp’s extraction solution preheated to 50 °C. The mixture was transferred quantitatively into a 100 mL volumetric flask, and the final volume was made up to the mark with additional warm extraction solution. The flask was then placed in a thermostatically controlled water bath maintained at 50 ± 1 °C for 1 h, with intermittent shaking every 10–15 min to ensure efficient extraction of soluble nitrogenous compounds. After incubation, the contents were immediately filtered through Whatman No. 40 filter paper to obtain a clear extract. A 10 mL aliquot of this filtrate was subsequently analyzed for soluble nitrogen using the semi–micro Kjeldahl method. Soluble nitrogen values were calculated and expressed as a percentage of sample weight, with all analyses performed in triplicate and reported as mean ± standard deviation.

Water activity

Water activity (a_w) of the Shrikhand samples was measured using a Rotronic Hygroskop Hygrolab-3 instrument equipped with an AW-DIO resistive electrolyte sensor (Rotronic AG, Switzerland).

Firmness

Compression testing to measure firmness was performed on a Lloyd LRX Plus (Lloyd Instruments, UK) texture analyzer fitted with a 50 N load cell. Shrikhand samples were prepared by tempering at 23 ± 1 °C for 30 min in an air-conditioned room (23 ± 1 °C, 55 ± 1% RH), and cylindrical cores (13 mm height, 78 mm diameter) were obtained using a cork borer to ensure uniform geometry. Each sample was placed centrally on the compression support plate and compressed using a flat plate probe at a crosshead speed of 50 mm/min to 50% deformation. Trigger force was set to 0.098 N. Force–time data were recorded and firmness was obtained.

Microbiological analysis

Probiotic counts were estimated following FSSAI method²⁴ using MRS agar for lactobacilli and Bifidobacterium Selective Count Agar (HiMedia Laboratories, Mumbai, Maharastra, India) for Bifidobacterium spp., with all plates overlaid with 4–5 mL of the same medium to maintain micro anaerobic conditions and incubated at 37 °C for 48 h prior to enumeration. Standard plate counts (SPC) were obtained by pour-plating appropriate dilutions on SPC Agar (HiMedia Laboratories, Mumbai, Maharastra, India) and incubating plates at 37 ± 0.5 °C for 48 h. Yeast and mould counts were carried out according to FSSAI method²⁴ using Potato Dextrose Agar (HiMedia Laboratories, Mumbai, Maharastra, India). Results for all microbial groups were expressed as log CFU/g, ensuring accurate characterization of microbial quality during storage.

Sensory evaluation

Sensory evaluation of both control and experimental Shrikhand samples was carried out by a trained panel. A panel of ten expert judges, each familiar with dairy product quality attributes, evaluated the samples using the BIS sensory scorecard²⁵. The scoring system allotted a maximum of 50 points for flavor, 35 points for body and texture, and 10 points for color and appearance. Since all samples were presented in identical, sealed polystyrene containers with lids, each sample automatically received the full packaging score of 5, ensuring uniformity in evaluation. Sensory assessments were performed for freshly prepared Shrikhand (day 0) as well as for samples stored for 7, 14, 21, 28, and 35 days under refrigerated conditions (7 ± 1 °C). To eliminate positional bias and expectancy effects, samples were coded using randomly generated three-digit numbers and presented in a randomized order. Evaluations were conducted in a controlled sensory laboratory equipped with neutral white lighting and ambient temperature maintained at 22 ± 1 °C. Each judge received samples individually and was instructed to cleanse the palate with potable water between evaluations to avoid sensory carryover. Panelists assessed each attribute independently based on standard descriptors for Shrikhand, including freshness and balance of sweetness–acidity for flavor, smoothness and firmness for body and texture, and brightness and uniformity for appearance²⁵.

Statistical analysis

All experimental data were subjected to statistical analysis using SPSS v.26 (IBM Corp., USA). Triplicate measurements were averaged and reported as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was employed to determine the significance of storage time on each quality parameter. When significant differences (p < 0.05) were found among storage intervals, Tukey’s HSD test was applied for post-hoc comparisons.

Results and discussions

pH and acidity (% LA) during storage

The acidity and pH of Shrikhand followed a clear and statistically significant pattern of post-fermentation evolution throughout the 35-day refrigerated storage period. Titratable acidity increased steadily from 0.98 ± 0.01% LA on day 0 to 1.33 ± 0.02% LA by day 35, while pH exhibited a corresponding decline from 4.37 ± 0.05 to 4.06 ± 0.01 over the same duration (Table 1). This inverse relationship between acidity and pH reflects the post-acidification behavior of fermented dairy products, driven largely by the continued but slow metabolic activity of lactic acid bacteria (LAB) at refrigeration temperatures (4 ± 1 °C).

Table 1.

Effect of storage period on the physico-chemical properties and firmness of Shrikhand.

Storage Period (days)	pH	Acidity (% LA)	FFA (µ eq. KOH/g)	Soluble Nitrogen (%)	Water Activity (aw)	Firmness (N)
0	4.37 ± 0.05ᵃ	0.98 ± 0.01ᶠ	0.64 ± 0.02ᶠ	0.24 ± 0.01ᶠ	0.9391 ± 0.002ᵃ	7.81 ± 0.01ᶠ
7	4.33 ± 0.01ᵃ	1.02 ± 0.01ᵉ	0.80 ± 0.01ᵉ	0.27 ± 0.03ᵉ	0.9378 ± 0.002ᵃ	8.00 ± 0.02ᵉ
14	4.23 ± 0.01b	1.08 ± 0.02ᵈ	0.97 ± 0.02ᵈ	0.34 ± 0.01ᵈ	0.9369 ± 0.002ᵃ	8.21 ± 0.02ᵈ
21	4.17 ± 0.03c	1.17 ± 0.02ᶜ	1.05 ± 0.02ᶜ	0.37 ± 0.01ᶜ	0.9355 ± 0.002ᵃ	8.45 ± 0.01ᶜ
28	4.12 ± 0.02d	1.23 ± 0.02ᵇ	1.09 ± 0.01ᵇ	0.41 ± 0.03ᵇ	0.9334 ± 0.002ᵃ	8.88 ± 0.01ᵇ
35	4.06 ± 0.01e	1.33 ± 0.02ᵃ	1.13 ± 0.03ᵃ	0.44 ± 0.01ᵃ	0.9330 ± 0.002ᵃ	9.23 ± 0.03ᵃ
SEm	0.016	0.007	0.009	0.009	0.001	0.009
CD (0.05)	0.05	0.022	0.026	0.029	0.003	0.027
CV (%)	0.95	1.512	1.815	5.385	0.183	0.207

Values represent mean ± standard deviation (n = 3). FFA = Free Fatty Acids; SEm = Standard Error of Mean; CD = Critical Difference; CV = Coefficient of Variation.

The ABY-3 culture comprising Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Bifidobacterium spp. is well known for its superior acidogenic potential^20,26,27. Among these, S. thermophilus and L. delbrueckii subsp. bulgaricus grows in symbiotic relationships. S. thermophilus produces formate and peptides that stimulate the metabolic activity of L. delbrueckii, which in turn releases amino acids and growth-promoting factors that further enhance S. thermophilus activity^20,26,27. Although this synergistic relationship slows under refrigeration, it does not cease entirely; thus, residual fermentation of lactose continues, gradually increasing acidity and reducing pH^20,26,27.

In addition to lactose metabolism, the incorporation of oat powder introduces soluble β-glucans, which possess viscosity-enhancing and water-binding properties that theoretically could moderate acidification by restricting lactose diffusion and altering the microenvironment around bacterial cells²⁸. Although this moderating effect may have contributed marginally to slowing acid production, the observed pH and acidity trends clearly indicate that the metabolic activity of the multi-strain culture remained sufficiently strong to drive a measurable and continuous acidification throughout storage^26–28.

From a product-quality standpoint, both acidity and pH remained within acceptable ranges for Shrikhand during the initial 28 days. However, by days 28–35, the cumulative increase in lactic acid and concurrent pH reduction suggest the development of a sharper acidic taste, potentially disrupting the characteristic sweetness–acidity balance that defines high-quality Shrikhand. Such late-stage acidification may also influence texture by promoting casein contraction and firmer gel structure.^26,27.

Free fatty acids (FFA) during storage

FFA concentrations showed a clear increasing pattern from the beginning to the end of storage. Starting at 0.64 ± 0.02 on day 0, FFA rose to 0.80 ± 0.01 at day 7, then increased to 0.97 ± 0.02 on day 14. By day 21, the value increased to 1.05 ± 0.02, followed by a slight increase to 1.09 ± 0.01 on day 28. The highest FFA level, 1.13 ± 0.03, was recorded at day 35 (Table 1). The increments were consistent across all intervals. This steady increase in FFA clearly indicates ongoing lipolytic activity within the product matrix. Lipolysis is a common biochemical event in fermented dairy products during refrigerated storage and occurs primarily due to enzymatic hydrolysis of milk fat triglycerides^29,30.

Multiple biochemical and microbiological mechanisms contributed to the observed increase in FFA values. The first major contributor is the activity of microbial lipases secreted by probiotic and starter cultures present in the ABY-3 system, including Lactobacillus acidophilus and Bifidobacterium spp^26,27. These organisms possess intracellular and extracellular lipolytic enzymes capable of hydrolyzing ester bonds in triglycerides even at low storage temperatures. Although the rate of enzymatic activity is reduced at 4 °C, it remains sufficient to steadily release short- and medium-chain fatty acids over time^26,27.

Another potential source of FFA is the presence of residual indigenous milk lipoprotein lipase (LPL), which, depending on the intensity of heat treatment applied to the milk, may not be fully inactivated. If any fraction of LPL remains active, even at low temperatures, it may continue cleaving milk fat globules, contributing to incremental increases in FFA during storage. Although milk was heated adequately, some residual enzyme activity cannot be fully excluded, particularly in high-fat systems such as Shrikhand^29,30.

Additionally, the incorporation of oat powder introduces small amounts of plant-derived lipids (typically 6–8% fat content)²⁸. These lipids, especially unsaturated fatty acids, may undergo mild oxidative or enzymatic breakdown, adding to the FFA. Despite the observed increase in FFA values, the levels remained moderate and within acceptable sensory scores. Excessive accumulation of FFA can lead to the development of rancid or soapy off-flavor due to the release of low-molecular-weight fatty acids such as butyric and caproic acids^29,30. However, the concentrations measured in this study did not approach levels associated with rancidity, suggesting that lipolytic activity was controlled and progressive rather than excessive.

Soluble nitrogen (%) during storage

Soluble nitrogen values also increased consistently from day 0 to day 35. The initial value of 0.24 ± 0.01 increased to 0.27 ± 0.03 at day 7 and increased further to 0.34 ± 0.01 at day 14. By day 21, soluble nitrogen reached 0.37 ± 0.01, increased slightly to 0.41 ± 0.03 at day 28, and attained the highest value of 0.44 ± 0.01 at day 35 (Table 1). This upward trend clearly indicates that protein degradation was continuously occurring within the Shrikhand matrix throughout the storage period. In fermented dairy systems, proteolysis is a natural and expected phenomenon, driven by a combination of microbial enzymatic activity and physicochemical interactions within the protein network³¹.

Several mechanisms contributed to the increase in soluble nitrogen. First, proteases produced by lactic acid bacteria present in the ABY-3 culture system, such as Lactobacillus acidophilus and Lactobacillus delbrueckii subsp. bulgaricus, play a central role in hydrolyzing casein molecules into peptides and free amino acids. These organisms possess efficient cell-envelope proteinases (CEPs) and intracellular peptidases that remain active even at refrigeration temperatures, albeit at a reduced rate^26–28. Second, autolysis of probiotic cells during prolonged storage releases intracellular enzymes into the Shrikhand matrix, further contributing to proteolysis. Autolytic breakdown is common in refrigerated fermented dairy products, particularly beyond 21–28 days, when cell viability gradually declines and cytoplasmic contents leak into the system^31–33. Third, starter culture metabolism may continue to drive limited but measurable hydrolysis of casein proteins. Even when microbial growth is slowed by cold temperatures, residual enzymatic activity persists. Finally, pH-induced modifications also contribute significantly to protein degradation. As storage progresses, the gradual decline in pH (from 4.37 to 4.06) destabilizes casein micelles, promoting demineralization, hydration, and partial disintegration of the protein matrix. This facilitates the release of nitrogenous compounds into the soluble fraction^31–33.

Water activity (a_w) during storage

Water activity (a_w) is a critical parameter governing the microbial, enzymatic, and physicochemical stability of fermented dairy products. The initial a_w of 0.9391 reduced slightly to 0.9378 on day 7 and continued decreasing slightly to 0.9369 on day 14. By day 21, a_w had declined to 0.9355, then to 0.9334 at day 28, and finally to 0.9330 at day 35 (Table 1). Although the absolute magnitude of this decrease appears modest, even minor reductions in a_w can exert considerable influence on the kinetics of biochemical reactions and overall product stability, particularly in semi-solid dairy matrices like Shrikhand.

The slight reduction in a_w value might be attributed to several complementary mechanisms. First, the incorporation of oat powder, which is rich in soluble β-glucans and dietary fibers, likely contributed to water binding^8–10. β-Glucans possess numerous hydroxyl groups capable of forming hydrogen bonds with water molecules, thereby converting some free water into a more tightly bound state. This effectively reduces the proportion of unbound water available to support microbial activity or participate in chemical reactions^8–10. Second, as storage progressed, casein micelles underwent acid-induced contraction and structural rearrangement due to the gradual decline in pH. This process often increases protein hydration, since shrinking micelles can expel loosely bound serum while simultaneously drawing in water into their internal matrix. This redistribution reduces the concentration of free water in the surrounding serum phase, which consequently slightly lowers a_w^34,35. Third, partial lactose crystallization may have occurred during extended storage, particularly under low-temperature conditions. Lactose crystallization traps water within the newly formed crystal lattice, further reducing some free water availability.

Firmness during storage

Firmness values increased clearly and steadily over the storage period. The initial firmness of 7.81 ± 0.01 increased to 8.00 ± 0.02 on day 7 and further to 8.21 ± 0.02 on day 14. By day 21, firmness reached 8.45 ± 0.01, then increased to 8.88 ± 0.01 on day 28. The highest value, 9.23 ± 0.03, was recorded at day 35 (Table 1). The increases were uniform and progressive across all time points. This post-fermentation increase in firmness is commonly observed in semi-solid fermented dairy products stored under refrigerated conditions.

Several interconnected biochemical and physicochemical mechanisms underpin the observed increase in firmness. The most influential factor is the acid-induced contraction of casein micelles³⁶. As titratable acidity increased and pH declined during storage, casein micelles approached their isoelectric point, leading to reduced electrostatic repulsion and enhanced hydrophobic interactions³⁶. This promotes the formation of a firm, more cohesive three-dimensional protein network that resists deformation, thereby increasing firmness³⁶.

The incorporation of oat powder also played a notable role in the rheological properties of the product. Oat powder contains soluble β-glucans, which possess strong water-binding and gel-forming capabilities. During the early days of storage, β-glucans progressively hydrate, swelling within the protein–fat matrix and increasing the viscosity of the product. As storage progresses, the hydration of β-glucans reaches equilibrium, contributing to a denser, more elastic matrix that enhances firmness^8,11,37. Moisture redistribution during storage further contributes to the changes in firmness of Shrikhand during storage. Water molecules shift from loosely bound regions to more strongly associated zones within the protein–fiber matrix. Such redistribution causes serum phase entrapment and reduces the mobility of water, a factor directly associated with firmness^8,11,37.

From a sensory and product stability perspective, the increase in firmness during the first 21–28 days may be desirable, as consumers often perceive a moderately firmer texture as richer and more satisfying. However, beyond this period, excessive firmness may negatively affect mouthfeel. By day 35, the firmness reached its highest value, suggesting the onset of over-firming, a typical indicator of aging and advanced post-acidification.

Microbiological quality

The microbiological quality of Shrikhand during refrigerated storage (7 ± 2 °C) is presented in Table 2. Significant changes (p < 0.05) were observed in probiotic counts, SPC, and yeast and mould counts over the 35-day storage period, reflecting both the biological activity of the probiotic culture and the influence of storage on microbial quality.

Table 2.

Effect of storage period on microbiological quality of Shrikhand.

Storage Period (days)	Probiotic Count (log CFU/g)	Standard Plate Count (log CFU/g)	Yeast and Mould Count (log CFU/g)
0	8.73 ± 0.02ᵃ	5.29 ± 0.01f	< 1
7	8.50 ± 0.05ᵇ	5.46 ± 0.01e	1.53 ± 0.22a
14	8.40 ± 0.05ᶜ	5.62 ± 0.06d	1.59 ± 0.20a
21	8.15 ± 0.04ᵈ	5.70 ± 0.01c	1.69 ± 0.27a
28	7.75 ± 0.03ᵉ	5.76 ± 0.05b	1.85 ± 0.27a
35	7.50 ± 0.06ᶠ	5.85 ± 0.05a	2.06 ± 0.16a
SEm	0.021	0.019	0.113
CD (0.05)	0.066	0.059	0.348
CV (%)	0.521	0.679	12.199

Probiotic count

The initial probiotic count of Shrikhand was 8.73 ± 0.02 log CFU/g on day 0, which gradually declined to 7.50 ± 0.06 log CFU/g by day 35 (Table 2). Although this reduction was statistically significant at each storage interval, the probiotic population consistently remained well above the recommended minimum threshold of 10⁶–10⁷ CFU/g, ensuring that the product retained its functional probiotic efficacy throughout the storage period. The gradual decline in probiotic viability can be attributed to a combination of several mechanisms. As storage progressed, increasing acidity and decreasing pH created a progressively harsh environment for probiotic organisms, particularly acid-sensitive species such as Lactobacillus acidophilus and Bifidobacterium spp., which tend to lose membrane integrity and enzymatic activity under low-pH conditions^38–40. In addition to acid stress, limited oxygen diffusion into the semi-solid Shrikhand likely contributed to viability loss, especially for Bifidobacterium spp., which are strict anaerobes and highly susceptible to oxidative exposure^38–40. Nutrient depletion also plays a role; probiotic cultures continue low-level metabolic activity under refrigeration, and the gradual exhaustion of fermentable lactose along with accumulation of metabolic by-products such as lactic acid and hydrogen peroxide reduces long-term cell survivability. Over extended refrigerated storage, natural cell senescence further accelerates viability decline due to oxidative stress, weakened membrane structure, and reduced ATP synthesis leading to autolysis^38–40. Despite these ongoing stressors, the probiotic count remained consistently above 7.5 log CFU/g until day 35, demonstrating the product’s ability to support probiotic survival and maintain its functional value throughout the assessed storage duration.

Standard plate count

The SPC of Shrikhand exhibited a gradual yet statistically significant increase over the 35-day refrigerated storage period, rising from 5.29 ± 0.01 log CFU/g on day 0 to 5.85 ± 0.05 log CFU/g by day 35 (Table 2). This progressive increase is characteristic of fermented dairy systems that contain other lactic acid–producing bacteria, as these organisms remain metabolically active even at low temperatures, which might be attributed to the observed increase in SPC.

Yeast and mould count

Yeast and mould counts in Shrikhand followed a gradual upward trend throughout the storage period, beginning from undetectable levels on day 0 and increasing to 1.53 ± 0.22 log CFU/g by day 7, 1.59 ± 0.20 log CFU/g on day 14, and reaching 2.06 ± 0.16 log CFU/g by day 35 (Table 2). Although the increases were statistically significant compared with the initial value, the progression was slow and remained within microbiologically acceptable limits for fermented dairy desserts during the entire evaluation period. The emergence of detectable yeast populations after day 7 is expected in dairy products with moderate acidity and high sugar content, as such conditions can support the growth of acid-tolerant, osmotolerant yeasts even under refrigeration. The steady rise observed from day 7 to day 35 suggests that the existing refrigeration and packaging conditions were effective at minimizing, though not completely inhibiting, yeast proliferation. However, the modest rise in yeast and mould counts observed reflects typical behavior of psychrotrophic and acid-tolerant microflora in refrigerated fermented products and suggests that the Shrikhand remained microbiologically acceptable and safe for consumption for at least 28–30 days of storage.

Sensory characteristics of shrikhand during storage

The sensory profile of Shrikhand was significantly influenced by the duration of refrigerated storage, as reflected by progressive declines in flavor, body and texture, color and appearance, and total sensory score (p < 0.05). At day 0, the product received a high overall acceptability score of 93.53 ± 0.78, indicating excellent sensory quality characterized by fresh flavor, smooth body and texture, and bright appearance. However, as storage advanced to 35 days, the total score decreased to 74.23 ± 0.70, demonstrating noticeable deterioration in overall sensory perception (Table 3).

Table 3.

Influence of storage period on the sensory characteristics of Shrikhand.

Storage Period (days)	Flavor (50)	Body and Texture (35)	Color and Appearance (10)	Total Score (95)
0	45.66 ± 0.58ᵃ	33.26 ± 0.12ᵃ	9.61 ± 0.22ᵃ	93.53 ± 0.78ᵃ
7	42.75 ± 0.08ᵇ	32.37 ± 0.24ᵇ	9.08 ± 0.16ᵇ	89.20 ± 0.32ᵇ
14	39.91 ± 0.09ᶜ	31.68 ± 0.24ᵇ	8.41 ± 0.20ᶜ	85.00 ± 0.09ᶜ
21	38.72 ± 0.03ᵈ	30.04 ± 0.62ᶜ	7.79 ± 0.13ᵈ	81.55 ± 0.51ᵈ
28	35.34 ± 0.19ᵉ	28.94 ± 0.59ᵈ	7.15 ± 0.13ᵉ	76.43 ± 0.70ᵉ
35	33.79 ± 0.07ᶠ	28.36 ± 0.80ᵈ	7.08 ± 0.05ᵉ	74.23 ± 0.70ᶠ
SEm	0.13	0.25	0.08	0.28
CD (0.05)	0.39	0.77	0.24	0.88
CV (%)	0.64	1.63	1.94	0.73

Values represent mean ± standard deviation (n = 3). FFA = Free Fatty Acids; SEm = Standard Error of Mean; CD = Critical Difference; CV = Coefficient of Variation.

Flavor scores gradually decreased across all storage intervals. The highest score was recorded on day 0 (45.66 ± 0.58), indicating maximum freshness and desirable taste immediately after preparation. By day 7, the score declined to 42.75 ± 0.08, followed by 39.91 ± 0.09 on day 14. Further reductions were observed at day 21 (38.72 ± 0.03) and day 28 (35.34 ± 0.19). The lowest flavor score was noted on day 35 (33.79 ± 0.07). Each interval showed a stepwise decrease in flavor acceptability (Fig. 1). This downward trend is primarily attributed to post-acidification, as evidenced by increasing titratable acidity and decreasing pH over the storage period. Increased acidity imparts an increasingly sharp, sour taste, which gradually diminishes the delicate sweetness–acidity balance characteristic of high-quality Shrikhand⁴.

Fig. 1.

Changes in sensory attributes of Shrikhand during storage.

Body and texture scores showed a similar downward trend, beginning with 33.26 ± 0.12 at day 0. The score reduced to 32.37 ± 0.24 at day 7 and remained statistically equal for day 14 (31.68 ± 0.24). A further drop was recorded at day 21 (30.04 ± 0.62), followed by 28.94 ± 0.59 at day 28 and 28.36 ± 0.80 at day 35 (Fig. 1). The decline in body and texture was gradual but consistent throughout the storage period. As storage progressed, the ongoing acidification caused casein micelles to contract and form a denser, more rigid gel network, leading to increased firmness and reduced spreadability. Although oat β-glucans in the formulation enhance water-binding capacity and contribute positively to texture in early storage, their continued hydration, combined with moisture redistribution within the matrix, ultimately results in a firmer, more compact structure^8–11.

Color and appearance scores decreased from an initial 9.61 ± 0.22 on day 0 to 9.08 ± 0.16 on day 7 and 8.41 ± 0.20 on day 14. By day 21, the score dropped further to 7.79 ± 0.13, continuing to 7.15 ± 0.13 on day 28 and 7.08 ± 0.05 on day 35 (Fig. 1). While the decline was less steep than flavor and texture, color and appearance still showed a noticeable downward progression. While color remained within acceptable limits throughout storage, subtle changes such as slight dulling or surface dehydration may have contributed to the decline.

Total sensory scores followed the same decreasing pattern observed in individual attributes (Table 3). The initial total score of 93.53 ± 0.78 decreased to 89.20 ± 0.32 on day 7 and 85.00 ± 0.09 on day 14. By day 21, the score declined to 81.55 ± 0.51, followed by 76.43 ± 0.70 at day 28. The lowest score was recorded on day 35 (74.23 ± 0.70). The decline remained steady across all intervals. During the first three weeks of storage, the product maintained high consumer appeal, with total scores remaining above 80 up to day 21. This indicates that the Shrikhand retained its desirable sensory attributes, balanced flavor, smooth and creamy body, and visually appealing appearance, throughout the early and mid-storage stages.

Beyond day 21, however, a more rapid decline in acceptability was observed. This accelerated deterioration can be attributed to several interrelated physicochemical and biochemical changes occurring within the product matrix. The most prominent contributors were the increasing acidity and associated decline in pH, which produced a sharper, more pronounced sourness that compensated the characteristic mild sweetness of Shrikhand.

Conclusion

The incorporation of 2% oat powder enriched with β-glucans considerably improved the structural stability, water-binding capacity, and functional quality of probiotic Shrikhand. Throughout 35 days of refrigerated storage, the product exhibited expected post-fermentation metabolic trends, including progressive increases in acidity, free fatty acids, and soluble nitrogen, yet maintained physicochemical characteristics within acceptable limits for at least 28 days. Importantly, probiotic viability remained above the minimum functional threshold (≥ 10⁷ CFU/g) during storage, confirming the suitability of oat β-glucans as supportive prebiotic components within dairy matrices. Sensory acceptability remained high for up to 21 days, after which intensified acidification and excessive firmness led to noticeable quality decline. Overall, the investigation demonstrated that oat powder incorporation offers dual technological and nutritional advantages for Shrikhand. These findings provide a strong scientific basis for developing clean-label, fiber-fortified, probiotic-rich Shrikhand with extended shelf-life. However, future studies should focus on inclusion of control formulation without oat powder to enable a more precise evaluation of the specific contribution of oat powder to the physicochemical, microbiological, and sensory properties of probiotic Shrikhand.

Author contributions

PS: Investigation, Data curation, Visualization, Writing – original draftAB: Conceptualization, Writing – Original draft, Writing – review and editing, Final approval of manuscriptSA: Validation, Critical review of manuscript ACP: Validation, Critical review of manuscript NDJ : Data curation, Validation.

Data availability

Data will be available based on request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical statement for sensory evaluation

Sensory evaluation was conducted in accordance with the ethical standards of Anand Agricultural University. The study protocol, including procedures for participant recruitment, informed consent, confidentiality, and safety, was reviewed and approved by the Institutional Ethics Committee, Anand Agricultural University. All panelists were adult volunteers affiliated with the university and were fully informed about the objectives and procedures prior to participation. Written informed consent was obtained from each participant. Sensory evaluation involved minimal risk, as samples were produced under strictly hygienic conditions. Participants were free to withdraw at any stage without penalty. No personal identifiers were collected, and all responses were anonymized to maintain confidentiality.