Received: 08/01/2026 Accepted: 21/03/2026 Published: 03/04/2026 1 of 8 https://doi.org/10.52973/rcfcv-e362895 Revista Cientíﬁca, FCV-LUZ / Vol. XXXVI ABSTRACT The survival of Lacticaseibacillus rhamnosus GG and two foodborne pathogens (Staphylococcus aureus and Escherichia coli) was monitored in L. rhamnosus GG – supplemented dairy milk and three plant-based alternatives (soy, almond, and coconut). Microbiological measurements were taken during fermentation (24 hours) and refrigerated storage (21 days, 4°C), using the L. rhamnosus GG – supplemented dairy group as the reference matrix for comparisons. Across all matrices, L. rhamnosus GG maintained counts above 7 log CFU·mL –1 throughout the experimental period. L. rhamnosus GG numbers did not differ signiﬁcantly between dairy and plant-based milks (P>0.05). Although time, milk type, and their interaction were statistically signiﬁcant (P<0.05), the observed shifts did not translate into a loss of L. rhamnosus GG culturability, and counts remained stable across milk types during storage. By day 21, S. aureus and E. coli counts were lower in almond and coconut milk than in soy and dairy milk (P<0.05). Overall, plant-based milk alternatives particularly almond and coconut appeared to be effective carriers for L. rhamnosus GG and performed comparably to dairy milk in sustaining probiotic viability. Because pathogen reduction was matrix-dependent, future work should examine underlying matrix-microbe interactions and validate these ﬁndings at industrial scale using integrated microbiological, physicochemical, and compositional analyses. Key words: Plant-based milk alternatives; Lacticaseibacillus rhamnosus GG; probiotic viability; pathogen inhibition; fermented functional beverages RESUMEN La supervivencia de Lacticaseibacillus rhamnosus GG y de dos patógenos transmitidos por alimentos (Staphylococcus aureus y Escherichia coli) se evaluó en leche láctea suplementada con L. rhamnosus GG y en tres alternativas vegetales a la leche (soja, almendra y coco). Las determinaciones microbiológicas se realizaron durante la fermentación (24 horas) y el almacenamiento refrigerado (21 días, 4°C), utilizando el grupo de leche de vaca suplementada con L. rhamnosus GG como matriz de referencia para la comparación. En todas las matrices, L. rhamnosus GG mantuvo recuentos superiores a 7 log UFC·mL –1 durante todo el período experimental. No se observaron diferencias signiﬁcativas en los recuentos de L. rhamnosus GG entre la leche de vaca y las leches de origen vegetal (P>0,05). Aunque el tiempo, el tipo de leche y su interacción mostraron efectos estadísticamente signiﬁcativos (P<0,05), estos cambios no se tradujeron en una pérdida de la viabilidad de L. rhamnosus GG, manteniéndose los recuentos estables en todos los tipos de leche durante el almacenamiento. Al día 21, los recuentos de S. aureus y E. coli fueron menores en las leches de almendra y coco en comparación con la leche de soja y la leche láctea (P<0,05). En conjunto, las alternativas vegetales a la leche en particular las de almendra y coco mostraron ser vehiculos eﬁcaces de L. rhamnosus GG y presentaron un desempeño comparable al de la leche de vaca en el mantenimiento de la viabilidad probiótica. Dado que la reducción de patógenos fue dependiente de la matriz, futuros estudios deberían profundizar en las interacciones matriz-microorganismo y validar estos hallazgos a escala industrial mediante análisis microbiológicos, ﬁsicoquímicos y composicionales integrados. Palabras clave: Alternativas a la leche de origen vegetal; Lacticaseibacillus rhamnosus GG; viabilidad probiótica; inhibición de patógenos; bebidas funcionales fermentadas Pathogen survival in dairy and plant-based probiotic fermented beverages containing Lacticaseibacillus rhamnosus GG Supervivencia de patógenos en bebidas fermentadas probióticas lácteas y de origen vegetal que contienen Lacticaseibacillus rhamnosus GG Esra Yılmaz Başar , Arife Ezgi Telli* Selcuk University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology. 42130, Konya, Türkiye. *Corresponding author: ezgiyilmaz@selcuk.edu.tr

Pathogen survival in dairy and plant-based probiotic fermented beverages / Yilmaz Başar and Telli __________________________________ 2 of 8 INTRODUCTION Plant-based milk alternatives such as soy (Glycine max), almond (Prunus dulcis), and coconut (Cocos nucifera) beverages have increasingly been explored as probiotic carriers, particularly in lactose-free food systems. These beverages are considered among the most popular alternatives due to their distinct nutrient proﬁles and potential prebiotic properties that support the survival and development of probiotics [1, 2]. Although research on probiotic viability in plant-based milk alternatives continues at an increasing pace, there is still a gap in understanding how these substrates directly affect the antimicrobial activity of probiotic bacteria against foodborne pathogens. While some studies highlight the supportive role of bioactive compounds in plant-based milk alternatives on probiotic survival, others report that the lack of milk-derived growth factors may compromise the culturable viability and functionality of probiotic strains through fermentation and storage [3, 4]. Pathogen contamination in plant based alternatives production can be caused by factors such as the microbial load of the raw material, inadequate pasteurization, unhygienic conditions during production, contamination from water and equipment, lack of employee hygiene, and improper storage processes [5, 6]. In these processes, Eschericha coli and Staphylococcus aureus are among the important pathogens that can cause contamination. S. aureus and E. coli are two of the most common bacterial pathogens responsible for foodborne illness worldwide [7]. Staphylococcus aureus is a Gram-positive bacterium capable of producing heat-stable enterotoxins that are one of the leading causes of food poisoning [8]. Similarly, E. coli , particularly pathogenic strains such as E. coli O157:H7, pose a signiﬁcant public health threat due to their ability to cause severe gastrointestinal infections that can result in hemorrhagic colitis and hemolytic uremic syndrome [9]. Several Lactobacillus species have been reported to inhibit foodborne pathogens through acidiﬁcation, metabolite production, and competitive interactions [10]. Previous studies have suggested that the type of milk substrate may influence the antimicrobial activity of probiotics, depending on its composition and physicochemical properties. The main objective of this experimental study was to evaluate the viability of Lacticaseibacillus rhamnosus GG (LbGG) in soy, coconut, almond, and dairy milk during a 24–hour fermentation period, followed by 21 days of storage at 4°C. Additionally, to assess the inhibitory effects of these probiotic-enriched beverages against S. aureus and E. coli during fermentation and storage, as well as to examine the impact of pH and acidity changes on probiotic viability and pathogen growth inhibition throughout this period. MATERIAL AND METHODS Experimental design and sample grouping The experimental setup was designed to investigate pathogen behavior in LbGG-supplemented milk alternatives. Commercially available soy milk, almond milk, coconut milk, and low fat dairy milk was obtained to be used in the production of probiotic-added plant-based beverages. The nutritional values of the milks used in the study are presented in TABLE I. Dairy milk served as the main control for comparing probiotic survival across milk types. Each milk type included three experimental groups: (i) LbGG + E. coli, (ii) LbGG + S. aureus, and (iii) LbGG-only control. Since all groups contained LbGG, the study does not isolate the probiotic’s direct inhibitory effect on pathogens. The study rather examines how pathogen viability varies among different milk types in the presence of LbGG. Additionally, LbGG survival was compared across plant based milk alternatives, with dairy milk acting as a reference. The experiment was conducted in triplicate at different time intervals. Inoculation of the microorganisms Prior to inoculation into plant-based milk alternatives, the bacterial cultures Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and LbGG ATCC 53103 were revived under appropriate conditions to ensure viability and optimal growth. E. coli and S. aureus were ﬁrst transferred into Tryptic Soy Broth (TSB) and incubated in a shaking incubator (Biosan ES–20, Türkiye) at 37°C for 24 hours (h), while LbGG was cultured in De Man, Rogosa, and Sharpe (MRS) broth at 37°C. The cultures were passaged back into fresh TSB to promote the exponential growth phase. The bacterial density of the ﬁnal cultures was determined using an optical density (OD) measurement (Allsheng-Nano–200, Deepak Biological House, India) at 600 nm (OD600) and adjusted to an OD600 value corresponding to ~10⁹ CFU·mL –1 [11]. The suspensions were then diluted to 10⁸ CFU·mL –1 prior to inoculation. Subsequently, 10 mL of the standardized culture (10⁸ CFU·mL –1 ) was added to 90 mL of each milk matrix, resulting in a ﬁnal inoculation level of approximately 10⁷ CFU·mL –1 . This ratio also aligns with regulatory standards requiring probiotics to remain above 10⁷ CFU·mL –1 . E. coli and S. aureus were inoculated at 10⁵ CFU·mL –1 to represent realistic contamination levels. Microbiological analysis Microbiological analyses were performed on designated for the fermentation and storage periods. Prior to sampling, each tube was gently inverted and homogenized by vortexing (Labart MVS–1), for 15 seconds (s). A 10 mL aliquot was taken from sterile glass bottles and mixed with 90 mL of Ringer’s solution. The mixture was homogenized using a stomacher for 90 s. Decimal dilutions (10 – ¹ TABLE I Basic nutritional composition of milk samples used in the study (100 mL) Nutrition Facts Dairy milk Soy milk Almond milk Coconut milk Energy 44 kcal 30 kcal 22 kcal 25 kcal Carbohydrate 4.7 g 2.4 g 2.4 g 2.7 g Protein 3.0 g 2.0 g 0.4 g 0.1 g Fat 1.5 g 1.3 g 1.1 g 1.5 g Salt 0.1 g 0.1 g 0.1 g 0.0 g

_______________________________________________________________________________________________Revista Cientiﬁca, FCV-LUZ / Vol. XXXVI 3 of 8 to 10 –10 ) were prepared by transferring 1 mL of the homogenate into 9 mL of Ringer’s solution in sterile tubes. Based on preliminary trials indicating high post-fermentation bacterial densities, extended dilution levels were included when necessary to ensure countable plates within the acceptable enumeration range. From each dilution, 100 µL was spreaded onto selective media. For the enumeration of LbGG de Man, MRS agar was used. The plates were incubated at 30°C for 24–48 h, and typical colonies were counted and expressed as log CFU·mL –1 . For E. coli enumeration, Tryptone Bile X-glucuronide agar was used. Plates were incubated at 37°C for 24 h, and green-blue, smooth, round colonies formed on the agar surface were counted and reported as log CFU·mL –1 . S. aureus was enumerated using Baird-Parker Agar. Plates were incubated aerobically at 37°C for 24–48 h, and black or grey-black colonies with a clear halo were counted and expressed as log CFU·mL –1 . Measurement of titratable acidity and pH In order to determine the acidity, 10 mL of milk samples were taken and transferred into flasks. 0,5 mL of 1 % phenolphthalein indicator were added to the milk. For titration, 0.1 N sodium hydroxide (NaOH) was added to the samples with a pipette until a light pink color was observed. When pink color was observed, the titration process was terminated and the amount of NaOH consumed was recorded. The titratable acidity was calculated as percent lactic acid using the following equation [12]. In this equation, 0.009 represents the amount (in grams) of lactic acid neutralized by 1 mL of 0.1 N NaOH; V is the volume (mL) of NaOH used during titration; and S is the volume (mL) of milk sample analyzed. A benchtop pH meter (WTW, inolab 720, Germany) was used for pH analysis. Statistical analysis Descriptive statistics were made for the measured milk samples and shown as Arithmetic Mean ± Standard Error. The conformity of the data to normal distribution was evaluated according to Shapiro Wilk results. For comparisons between milk types and time points, the General Linear Model was used, as the same samples were measured at multiple time intervals (0, 7, 14, and 21 d for microbiological data; 0, 8, 24 h and d 0, 7, 14, 21 for pH and acidity). The Bonferroni post-hoc tes was applied to determine pairwise differences between groups. All analyses were performed using the SPSS Statistics version 22.0 software (IBM Corp., Armonk, NY, USA). A P-value less than 0.05 was considered statistically signiﬁcant. RESULTS AND DISCUSSION Viability of Lacticaseibacillus rhamnosus GG in different milk types during storage In all milk types, LbGG exhibited a marked increase after 24 h of fermentation compared to the initial inoculation level (d 0), reaching high-density populations exceeding 9.5 log₁₀ CFU·mL –1 . Throughout the 21 d refrigerated storage period at 4°C, LbGG maintained viable counts well above the functional probiotic threshold of 7 log₁₀ CFU·mL –1 (TABLE II). Given that dairy milk served as the reference matrix and experimental conditions were standardized across treatments, the sustained high-density viability of LbGG in almond, coconut, and soy milks indicates that plant-based alternatives can effectively support probiotic maintenance under the tested conditions. LbGG viability signiﬁcantly increased during the ﬁrst 24 h of fermentation. Following the initial increase during fermentation, LbGG reached high-density populations (≥ 9 log₁₀ CFU·mL –1 ) and remained at elevated viability levels throughout storage (d 7, 14, and 21). Although exact quantiﬁcation at very high cell densities was limited by plate count constraints, viable counts consistently remained well above the functional probiotic threshold (> 7 log₁₀ CFU·mL –1 ) during refrigerated storage. Similar trends have been reported in fermented plant-based beverages. Kılınç et al. [13] observed that several probiotic strains, including S. thermophilus, L. delbrueckii subsp. bulgaricus, and L. acidophilus, maintained viability above 6 log₁₀ CFU·mL –1 during 21 d of storage at 4°C. Comparable stability has also been documented for LbGG grown in various non-dairy substrates [6, 11, 14], supporting the sustained viability observed in the present study. Survival of Staphylococcus aureus and Escherichia coli in Lacticaseibacillus rhamnosus GG-containing milk samples An interaction was observed between time and milk type (P<0.001), indicating that the survival of S. aureus varied depending on the milk matrix. While S.aureus counts remained relatively high in dairy and soy milk throughout storage, lower counts were detected in almond and coconut milk. S. aureus was not detectable in coconut milk by d 21, which may suggest a possible inhibitory effect associated with this matrix (TABLE III). The survival of E. coli was influenced by the type of milk used (P<0.001), suggesting that differences in milk composition may have affected the viability of the pathogen during storage. Although E. coli observed to have high viability in dairy and soy milk, a more noticeable decrease in viability was observed in coconut and almond milk samples. By d 21, E. coli counts in these plant-based milks had declined to approximately 4 log₁₀ CFU·mL –1 , indicating a relatively lower persistence compared to other milk types (TABLE IV, FIG. 1.). TABLE II Viability of Lacticaseibacillus rhamnosus GG in diﬀerent milk types over time (log₁₀ CFU·mL –1 ) Milk Type Day 0 Day 1* Day 7 Day 14 Day 21 Dairy milk 6.76 ± 0.00 > 9.5 > 9.5 > 9.5 > 9.5 Soy milk 7.08 ± 0.10 > 9.5 > 9.5 > 9.5 > 9.5 Coconut milk 7.11 ± 0.28 > 9.5 > 9.5 > 9.5 > 9.5 Almond milk 6.84 ± 0.31 > 9.5 > 9.5 > 9.5 > 9.5 *: 24 fermentation

Pathogen survival in dairy and plant-based probiotic fermented beverages / Yilmaz Başar and Telli __________________________________ 4 of 8 Greater reductions in pathogen culturability were observed in coconut and almond milks compared to dairy and soy, which may be influenced by differences in matrix composition. Although the current study did not analyze the speciﬁc compositional features of the milk matrices, previous research has reported that almond and coconut milks may contain medium-chain fatty acids and polyphenolic compounds with antimicrobial activity [15, 16]. It has been shown that polyphenols extracted from almond skins exhibit antimicrobial activity against foodborne pathogens [17]. Paul et al. [1] emphasized that soy, coconut, and almond milks contain bioactive compounds like phenolic acids and natural ﬁbers that contribute improved microbial viability during fermentation and storage. The high linoleic and α–linolenic acid content of almond milk may also decrease the viable count of S. aureus that decreased signiﬁcantly after d 1 (P<0.05), while a signiﬁcant reduction in E. coli was observed only on d 21 (P<0.05). Taken together linoleic acid present in almond milk, may be considered one of the contributors that might exert an inhibitory effect on both S. aureus and E. coli. Coconut milk is a rich source of medium-chain fatty acids, particularly lauric acid, and also contains amino acids such as L-arginine [18]. The lauric acid and its monoglyceride form monolaurin, are known for their antibacterial properties, especially against Gram-positive pathogens [19]. The high content of these fatty acids may have contributed to the inhibition of S. aureus observed exclusively in coconut milk in the present study. In this context, Shori and Zahrani [20] have also reported that the ﬁber and sugar content of plant-based milks plays a key role in promoting probiotic growth and supporting their survival during refrigerated storage. These compounds may have synergistic effect to enhance the nutritional and functional properties of plant- based probiotic beverages. Jogi et al. [21] similarly evaluated the antimicrobial activity of probiotic strains isolated from plant-based milks and reported that L. plantarum, L. acidophilus, L. rhamnosus, L. casei, L. lactis, and B. longum exhibited antagonistic activity against E. coli, Shigella, Salmonella, and S. aureus in almond, coconut, and soy milks. Their ﬁndings also showed that milk composition can influence both probiotic viability and pathogen inhibition. In this regard, fermentation conditions and the speciﬁc microbial interactions during the fermentation process found to be attributable in determining the inhibitory effect in this study. TABLE III Survival of Staphylococcus aureus in Lacticaseibacillus rhamnosus GG-containing milk samples S. aureus (log CFU·mL –1 ) Sampling Time (days) P–value Day 0 Day 1 Day 7 Day 14 Day 21 Time Milk Time*milk Dairy milk 4.67 ± 0.40 a,A 6.58 ± 0.23 b,C 7.19 ± 0.25 c,D 6.04 ± 0.18 c,B 5.36 ± 0.17 c,A < 0.001 < 0.001 < 0.001 Soy milk 4.55 ± 0.06 a,A 7.53 ± 0.04 c,C 7.66 ± 0.78 d,C 6.28 ± 0.08 c,B 5.14 ± 0,27 c,A Coconut milk 4,70 ± 0,21 a,D 6.07 ± 0.11 a,E 3.15 ± 0.08 a,C 1.64 ± 0.06 a,B 0.00 ± 0.00 a,A Almond milk 4,34 ± 0,36 a,B 6.46 ± 0.19 ab,D 5.12 ± 0.04 b,C 3.89 ± 0.26 b,B 3.34 ± 0.35 b,A The letters a, b, c, d in the table are used to express the diﬀerences between rows; the letters A, B, C, D, E are used to express the diﬀerences between columns. Diﬀerent letters in the same column/row are signiﬁcant (P<0.05) TABLE IV Survival of Escherichia coli in Lacticaseibacillus rhamnosus GG-containing milk samples E. coli (log CFU·mL –1 ) Sampling Time (days) P–value Day 0 Day 1 Day 7 Day 14 Day 21 Time Milk Time*milk Dairy milk 4.57 ± 0.06 a,A 9.64 ± 0.10 c,C 9.48 ± 0.45 b,C 9.61 ± 0.29 b,C 8.13 ± 0.23 b,B < 0.001 < 0.001 < 0.001 Soy milk 4.73 ± 0.14 a,A 8.81 ± 0.43 b,BC 8.95 ± 0.23 b,C 9.69 ± 0.37 b,C 8.21 ± 0.23 b,B Coconut milk 4.64 ± 0.18 a,B 7.64 ± 0.05 a,C 7.41 ± 0.28 a,C 7.31 ± 0.35 a,C 3.87 ± 0.29 a,A Almond milk 4.65 ± 0.12 a,B 7.77 ± 0.22 a,C 7.59 ± 0.14 a,C 7.46 ± 0.31 a,C 3.91 ± 0.50 a,A The letters a, b, c, d in the table are used to express the diﬀerences between rows; the letters A, B, C, D, E are used to express the diﬀerences between columns. Diﬀerent letters in the same column/row are signiﬁcant (P<0.05) FIGURE 1. Pathogen reduction rates in diﬀerent milk types

_______________________________________________________________________________________________Revista Cientiﬁca, FCV-LUZ / Vol. XXXVI 5 of 8 Similar results by the same researchers, Zahrani and Shori [2] compared probiotic viability and antioxidant activity in soy and almond milks fermented with LbGG. The researchers observed that the strains maintained their viability during 21 d of storage consistent with the ﬁndings of the present study. They emphasized the role of synergistic matrix effects in supporting probiotic functionality. The initial pH levels differed among milk types prior to LbGG inoculation (P<0.05). After 24 h of fermentation, the lowest pH value was measured in dairy milk (4.62 ± 0.02), indicating greater acidiﬁcation compared to the others. During the cold storage period (0–21 d), pH values either continued to decrease or remained relatively stable, depending on the milk type. Almond milk showed the highest decrease in pH, reaching 4.31 ± 0.02 by d 21. In contrast, soy milk retained higher pH values throughout storage, ending at 4.97 ± 0.02. These results suggest that the extent of acidiﬁcation during storage is influenced by both the milk matrix and the post-fermentation pH level (TABLE V). The acidity values were analyzed before fermentation; the dairy milk was measured at the highest value (P<0.05). The acidity increased signiﬁcantly on the last d of storage (d 21) compared to the ﬁrst measurement (d 0) in all milk samples except soy milk. In soy milk, an increase was ﬁrst observed for acidity after the d 0 measurement, and no change was observed on the 7 th and 14 th d of storage. On d 21, it decreased and reached a level lower than the d 0 measurement (TABLE VI). Beyond the influence of milk composition, the extended fermentation period in this study (24 h) may have also contributed to the observed microbial behavior and metabolite production. During extended fermentation periods, LAB typically enter a stationary growth phase, which enables the activation of secondary metabolic pathways and the onset of mixed acid fermentation [22]. In a study by Montemurro et al. [23], the production of soy-based yogurt through a 6 hour fermentation with LAB resulted in lower levels of digestible protein compared to unfermented soy milk. This was attributed to the weak proteolytic activity during the early stages of fermentation, when LAB preferentially utilize free amino acids available in the substrate. Yuyama et al. [24] tested the antibioﬁlm effects of various fatty acids, including linoleic acid. They reported that Gram-positive bacteria were more susceptible to the inhibitory effects of long- chain fatty acids compared to Gram-negative bacteria. Similarly, Kusumah et al. [25] demonstrated that both linoleic and α–linolenic acids exhibited antibacterial activity against S. aureus and Bacillus subtilis, signiﬁcantly impairing bacterial growth. In this study, a similar mechanism might also explain the antimicrobial effect observed in coconut milk, which compared to dairy milk showed greater suppression of S. aureus and is characterized by its distinct medium-chain fatty acid proﬁle. Considering that the fermentation period in the present study was extended to 24 h, it is possible that proteolytic activity may have increased following the initial depletion of free amino acids. The prolonged fermentation may allow for more extensive protein degradation and secondary metabolite production. Compared to dairy milk, the observed trends in plant-based alternatives suggest that under equivalent fermentation conditions, they can support similar microbial activity and metabolite development. When compared with dairy milk, plant-based milks particularly almond and coconut showed more pronounced pH declines by the end of storage, implying comparable fermentation dynamics. But in contrast, Demircan [26] reported a lower post-fermentation pH TABLE V pH Values of milk samples during fermentation and cold storage Milk Type 0 h (F) 8 h (F) 24 h (F) Day 0 (S) Day 7 (S) Day 14 (S) Day 21 (S) Dairy milk 6.84 ± 0.02 b,A 6.78 ± 0.02 c,B 4.62 ± 0.02 a,C 4.62 ± 0.02 a,C 4.51 ± 0.02 a,B 4.46 ± 0.03 b,A 4.61 ± 0.02 b,C Soy milk 7.26 ± 0.01 c,A 6.62 ± 0.02 b,B 5.65 ± 0.02 c,C 5.65 ± 0.02 c,C 5.20 ± 0.03 c,B 4.99 ± 0.02 c,A 4.97 ± 0.02 c,A Coconut milk 6.73 ± 0.01 a,A 6.27 ± 0.03 a,B 4.77 ± 0.07 b,C 4.77 ± 0.07 b,C 4.56 ± 0.01 b,B 4.50 ± 0.01 b,A 4.61 ± 0.01 b,B Almond milk 7.65 ± 0.02 d,A 6.91 ± 0.02 d,B 4.66 ± 0.02 ab,C 4.66 ± 0.02 a,C 4.48 ± 0.02 a,B 4.35 ± 0.02 a,A 4.31 ± 0.02 a,A F: fermentation, S: storage. Diﬀerent lowercase letters (a, b, c, d) within rows indicate signiﬁcant diﬀerences over time (P<0.05). Uppercase letters (A, B, C, D) within columns indicate diﬀerences between milk types at each time point TABLE VI Titratable acidity values of milk samples during fermentation and cold storage Milk Type 0 h (F ) 8 h (F) 24 h (F) Day 0 (S) Day 7 (S) Day 14 (S) Day 21 (S) Dairy milk 0.209± 0.002 d,A 0.230 ± 0.004 d,B 0.261 ± 0.004 c,C 0.261 ± 0.004 c,A 0.430 ± 0.002 c,B 0.566 ± 0.000 d,C 0.713 ± 0.007 b,BC Soy milk 0.053± 0.003 c,A 0.120 ± 0.002 c,B 0.140 ± 0.004 b,C 0.140 ± 0.004 b,A 0.162 ± 0.005 b,B 0.163 ± 0.004 c,B 0.120 ± 0.002 a,AB Coconut milk 0.022± 0.000 b,A 0.067 ± 0.004 b,B 0.066 ± 0.003 a,B 0.066 ± 0.003 a,A 0.079 ± 0.002 a,B 0.070 ± 0.002 a,AB 0.274 ± 0.352 ab,AB Almond milk 0.009± 0.00 a,A 0.049 ± 0.001 a,B 0.058 ± 0.003 a,B 0.058 ± 0.003 a,A 0.073 ± 0.004 a,B 0.101 ± 0.002 b,C 0.129 ± 0.003 a,ABC F: fermentation, S: storage. Diﬀerent lowercase letters (a, b, c, d) within rows indicate signiﬁcant diﬀerences over time (P<0.05). Uppercase letters (A, B, C, D, E) within columns indicate diﬀerences between milk types at each time point

Pathogen survival in dairy and plant-based probiotic fermented beverages / Yilmaz Başar and Telli __________________________________ 6 of 8 of 4.2 in soy milk fermented with LAB at 37°C for 6 h. Variations in pH and acidity observed appear to be influenced by factors such as the type of probiotic strains used, fermentation duration, and the composition of plant-based milks. A similar study by Deziderio et al. [27] reported minimal changes in pH and acidity in almond and soy milk after 12 h of fermentation. In contrast, the present study resulted in more pronounced decreases in pH and increases in titratable acidity. The prolonged fermentation period may have been the difference between studies that highligting the importance of fermentation time in physicochemical properties. Erik and Ergenekon [28] observed a gradual increase in pH and a decrease in acidity in whey cheese during storage, whereas the opposite trend was noted in probiotic milk samples in this study. These discrepancies could be influenced by differences in milk types and product type. The pH of soy milk after 24 h of fermentation was higher than that of cow milk (P<0.05), highlighting a distinct buffering response. Similarly, Okur [29] reported higher pH values in soy milk-based beverages, which were attributed to the high buffering capacity of soy milk. The buffering capacity may influence fermentation dynamics and acidiﬁcation behavior. It is known as a key factor affecting changes in acidity and pH. The protein content and intrinsic pH level of the food are primary determinants of its buffering potential. In a related study, Wang et al. [30] fermented dairy milk, soy milk, and oat milk until their pH values reached 4.5. Although the initial pH of dairy milk was higher than that of soy milk, it reached the target pH more rapidly than soy milk, suggesting that dairy milk had a lower buffering capacity under the same conditions. The more pronounced acidiﬁcation observed in dairy milk may be partially explained by the presence of lactose, a readily fermentable carbohydrate efﬁciently utilized by lactic acid bacteria such as LbGG, whereas plant-based alternatives lack lactose and differ in carbohydrate composition. This relationship between protein content and buffering capacity has also been linked to fermentation duration in various plant- based milks. Masia et al. [31] stated that plant-based milks containing high-molecular-weight proteins exhibit stronger buffering capacities, resulting in prolonged fermentation times. Consistent with this higher protein content and buffering potential, soy milk maintained a higher pH value after 24 h of fermentation compared to dairy milk [32], suggesting that it resisted acidiﬁcation more effectively under similar conditions. A limitation of this study is the absence of pathogen-only controls within each milk matrix. Therefore, the observed reductions should be interpreted as matrix-associated effects in LbGG-containing systems rather than being attributed exclusively to probiotic activity. Future studies may further clarify the relative contribution of matrix composition and microbial interactions. CONCLUSION The reductions observed in S. aureus and E. coli counts in almond and coconut milks may result from a combined effect of LbGG activity and matrix-related characteristics. Although this study did not aim to isolate the speciﬁc contribution of each factor, the results provide a meaningful basis for future research focusing on targeted probiotic-pathogen-matrix interactions. Overall, the ﬁndings highlight that certain plant-based milk alternatives may serve as effective probiotic carriers, offering functional potential similar to dairy milk. Further studies are needed incorporating detailed compositional, nutritional, and sensory evaluations to clarify the mechanisms underlying these effects and support the development of optimized non-dairy probiotic beverages. ACKNOWLEDGEMENTS This study was financially supported by Selcuk University Scientific Research Projects Coordination Unit under project number 24202005. Conflicts of Interest The authors declare no conflicts of interest. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by both authors. The ﬁrst draft of the manuscript was written by EYB and all authors commented on previous versions of the manuscript. All authors read and approved the ﬁnal manuscript. Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors. Data availability The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. BIBLIOGRAPHICS REFERENCES [1] Paul AA, Kumar S, Kumar V, Sharma R. Milk Analog: Plant based alternatives to conventional milk, production, potential and health concerns. Crit. Rev. Food Sci. Nutr. [Internet]. 2020; 60(18):3005–3023. doi: https://doi.org/gj33hn [2] Zahrani AJA, Shori AB. Viability of probiotics and antioxidant activity of soy and almond milk fermented with selected strains of probiotic Lactobacillus spp. LWT 2023; 176:114531. doi: https://doi.org/g6jmr8 [3] Ranadheera RDCS, Baines SK, Adams MC. Importance of food in probiotic efﬁcacy. Food Res. Int. [Internet]. 2010; 43(1):1–7. doi: https://doi.org/cwcsx8 [4] Craig WJ, Fresán U. International analysis of the nutritional content and a review of health beneﬁts of non-dairy plant- based beverages. Nutrients [Internet]. 2021; 13(3): 842. doi: https://doi.org/gqzs25

_______________________________________________________________________________________________Revista Cientiﬁca, FCV-LUZ / Vol. XXXVI 7 of 8 [5] Kyrylenko A, Eijlander RT, Alliney G, Lucas-van de Bos E, Wells- Bennik MHJ. Levels and types of microbial contaminants in different plant-based ingredients used in dairy alternatives. Int. J. Food Microbiol. [Internet]. 2023; 407:110392. doi: https://doi.org/gszqzb [6] Rasika DMD, Vidanarachchi JK, Luiz SF, Azeredo DRP, Cruz AG, Ranadheera CS. Probiotic delivery through non-dairy plant- based food matrices. Agriculture [Internet]. 2021; 11(7):599. doi: https://doi.org/qx29 [7] Abebe E, Gugsa G, Ahmed M. Review on major food–borne zoonotic bacterial pathogens. J. Trop. Med. [Internet]. 2020; 2020:4674235. doi: https://doi.org/gmq43k [8] Fetsch A, Johler S. Staphylococcus aureus as a foodborne pathogen. Curr. Clin. Microbiol. Rep. [Internet]. 2018; 5:88– 96. doi: https://doi.org/qx3b [9] Lorenzo JM, Munekata PE, Dominguez-Valencia R, Pateiro M, Saraiva JA, Franco D. Main groups of microorganisms of relevance for food safety and stability: General aspects and overall description. In: Barba FJ, Santana AS, Orlien V, Koubaa M, editors. Innovative technologies for food preservation. London (UK): Academic Press; 2018 [cited 20 Dec 2025]. p. 53–107. Available in: https://goo.su/H0F4 [10] Yeşilova Y, Sula B, Yavuz E, Uçmak D. Probiyotikler [Probiotics]. J. Kartal TR. 2010; 21(1):49–56. https://goo.su/EsJ6t [11] Tomasello F, Valero A, Serraino A, Possas A. Methods of inoculation and quantiﬁcation for collecting data on microbial responses in foods. In: Alvarenga VO, editor. Basic protocols in predictive food microbiology. New York (USA): Springer; 2023. p. 31–46. doi: https://doi.org/qx7z [12] Tekinşen OC, Atasever M, Keleş A, Tekinşen KK. Süt, Yoğurt, Tereyağı, Peynir: Üretim control [Milk, Yogurt, Butter, Cheese: Production control]. Konya (Türkiye): Selçuk University Press; 2002. 91 p. Turkish. [13] Kılınç GE, Keser A, Özer HB. Determination of nutritional value, antioxidant activities, microbiological and sensory properties of almond, soy and oat based fermented beverages. J. Culin. Sci. Technol. [Internet]. 2026; 24(1):16–32. doi: https://doi.org/qx75 [14] Fatima SM, Hekmat S. Microbial and sensory analysis of soy and dairy milk-based yogurt as a probiotic matrix for Lactobacillus rhamnosus GR–1. Fermentation [Internet]. 2020; 6(3):74. doi: https://doi.org/qx76 [15] Bouarab-Chibane L, Degraeve P, Ferhout H, Bouajila J, Oulahal N. Plant antimicrobial polyphenols as potential natural food preservatives. J. Sci. Food Agric. [Internet]. 2019; 99(4):1457–1474. doi: https://doi.org/gkf5jt [16] Nitbani FO, Tjitda PJP, Nitti F, Jumina J, Detha AIR. Antimicrobial properties of lauric acid and monolaurin in virgin coconut oil: a review. ChemBioEng. Rev. [Internet]. 2022; 9(5):442–461. doi: https://doi.org/qx78 [17] Mandalari G, Bisignano C, D’Arrigo M, Ginestra G, Arena A, Tomaino A, Wickham MSJ. Antimicrobial potential of polyphenols extracted from almond skins. Lett. Appl. Microbiol. [Internet]. 2010; 51(1):83–89. doi: https://doi.org/d3kk9r [18] Eyres L, Eyres MF, Chisholm A, Brown RC. Coconut oil consumption and cardiovascular risk factors in humans. Nutr. Rev. [Internet]. 2016; 74(4):267–280. doi: https://doi.org/f8jfvd [19] Nasir NAMM, Abllah Z, Jalaludin AA, Shahdan IA, Abd-Manan WNHW. Virgin coconut oil and its antimicrobial properties against pathogenic microorganisms: a review. Adv. Health Sci. Res. [Internet]. 2018 [cited 13 Oct 2025]; 192–199. Available in: https://goo.su/ByKb [20] Shori AB, Al Zahrani AJ. Non-dairy plant-based milk products as alternatives to conventional dairy products for delivering probiotics. Food Sci. Technol. [Internet]. 2022; 42:e101321. doi: https://doi.org/qx8c [21] Jogi S, Adhao A, Pachori RR. Exploration of probiotic bacteria from plant based milk. Int. J. Res. Anal. Rev. [Internet]. 2024 [cited 22 Dec 2025]; 11(1):157–163. Available in: https:// goo.su/xuUSjt [22] Genet BML, Xiao H, Christensen LF, Laforce IN, Mohammadifar MA, Bang-Berthelsen CH, Hansen EB. Selection of proteolytic LAB starter cultures for acidiﬁcation of soy based dairy alternatives. LWT [Internet]. 2023; 184:115082. doi: https://doi.org/qx8h [23] Montemurro M, Pontonio E, Coda R, Rizzello CG. Plant-based alternatives to yogurt: state-of-the-art and perspectives of new biotechnological challenges. Foods. [Internet]. 2021; 10(2):316. doi: https://doi.org/gt57q2 [24] Yuyama KT, Rohde M, Molinari G, Stadler M, Abraham WR. Unsaturated fatty acids control bioﬁlm formation of Staphylococcus aureus and other gram-positive bacteria. Antibiotics 2020; 9(11):788. doi: https://doi.org/qx8k [25] Kusumah D, Wakui M, Murakami M, Xie X, Yukihito K, Maeda I. Linoleic acid, α–linolenic acid, and monolinolenins as antibacterial substances in the heat-processed soybean fermented with Rhizopus oligosporus. Biosci. Biotechnol. Biochem. [Internet]. 2020; 84(6):1285–1290. doi: https://doi.org/qx8m [26] Demircan B. Probiyotik laktik asit bakterileri kullanılarak bitkisel süt ikamelerinden fermente içecek tasarımı [Production of novel vegetal based beverages fermented by probiotic lactic acid bacteria]. [master’s thesis on the Internet]. İstanbul (Türkiye): Istanbul Technical University; 2017 [cited 20 Jan 2026]. 99 p. Turkish. Available in: https://goo.su/Cd2kx [27] Deziderio MA, de Souza HF, Kamimura ES, Petrus RR. Plant-based fermented beverages: Development and characterization. Foods [Internet]. 2023; 12(22):4128. doi: https://doi.org/qx8n [28] Erik S, Ergenekon FS. Lor Peynirlerine İlave Edilen Probiyotik Kültürlerin Bazı Patojen Mikroorganizmaların Yaşam Süreleri Üzerine Etkisinin Araştırılması [Investigation of the effect of probiotic cultures added in lor cheese on the life span of some pathogenic microorganisms]. Akademik Et ve Süt Kurumu Derg. [Internet]. 2024 [cited 20 Dec 2025]; (7):4–14. Turkish. Available in: https://goo.su/ETJn3A8 [29] Okur HH. Probiyotik Lactobacillus rhamnosus GG (ATCC 53103) suşu kullanarak fermente süt ürünü üretimi [Production of fermented milk product by using probiotic Lactobacillus rhamnosus GG (ATCC 53103) strain]. [master’s thesis on the Internet]. İzmir (Türkiye): Ege University; 2022 [Cited 20 Jan 2026]. 114 p. Turkish. Available in: https://goo.su/XnFVno8

Pathogen survival in dairy and plant-based probiotic fermented beverages / Yilmaz Başar and Telli __________________________________ 8 of 8 [30] Wang X, Kong X, Zhang C, Hua Y, Chen Y, Li X. Comparison of physicochemical properties and volatile flavor compounds of plant-based yoghurt and dairy yoghurt. Food Res. Int. [Internet]. 2023; 164:112375. doi: https://doi.org/grs87k [31] Masia C, Geppel A, Jensen PE, Buldo P. Effect of Lactobacillus rhamnosus on Physicochemical Properties of Fermented Plant-Based Raw Materials. Foods [Internet]. 2021; 10(3):573. doi: https://doi.org/gjp38f [32] Yiğit AA. Animal and plant-based milk and their antioxidant properties. MAEU Vet. Fak. Derg. [Internet]. 2019; 4(2):113– 122. doi: https://doi.org/qx83