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Full Research Article

Efficacy of Tulsi (Ocimum sanctum) Extract for Preserving Raw Milk in the Informal Marketing System of Bangladesh Across Different Seasons 

T
Tanni Chanda1,*
A
Arifur Rahman1
I
Iftakher Alam Sarkar2
N
Naeem Hossain1
G
Goutam Kumar Debnath3
1Department of Dairy Science, Faculty of Animal Science and Veterinary Medicine, Patuakhali Science and Technology University, Barisal 8602, Bangladesh.
2Buffalo Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341, Bangladesh.
3Department of Dairy and Poultry Science, Faculty of Veterinary Medicine, Chattogram Veterinary and Animal Sciences University. Chattogram 4225, Bangladesh.

ABSTRACT

Background: Like other Southeast Asian countries, Bangladesh’s informal milk market frequently faces issues of contamination due to its high perishability and inadequate cooling facilities. To resolve this issue, this study investigated the use of Tulsi (Ocimum sanctum) leaf extract in different concentrations as a natural and secure preservative.

Methods: In this study, we included a control group and applied concentrations of 0.5%, 1% and 1.5% aqueous Tulsi leaf extract to raw milk, monitoring their effects over six hours at two-hour intervals across summer, rainy and winter seasons. The sampling was conducted in the Babuganj area of Barisal, Bangladesh, from July 2022 to June 2023. Different parameters, such as pH level, percent (%) of acidity and microbial count of different samples, were measured at the Dairy lab. of the Faculty of Animal Sciences and Veterinary Medicine of Patuakhali Science and Technology University.

Result: The 1.5% Tulsi extract (T2) was found to be most effective in maintaining pH stability, controlling acidity and microbial load. However, this higher concentration (T3) caused a noticeable color change in the milk. Conversely, the lower 0.5% concentration (T1) demonstrated lower effectiveness in controlling microbial growth (DMC). The 1% concentration (T2) is identified as a better alternative to synthetic preservatives, balancing efficacy with minimal sensory alteration and controlling microbial activity. Further research is needed on its long-term effects.

INTRODUCTION

Dairy is an emerging sector in Bangladesh with significant potential to contribute substantially to economic growth. Historically, Bangladesh has been an agricultural country, and the lives of Bangladeshi people are closely related to agriculture. Dairying is a principal part of Agriculture that has an intimate relation to livelihood, especially in rural areas. Over time, the features of dairying have been transformed from a rural livelihood to a highly profitable industry. The number of cattle populations is now 25.01 million, with milk production of 15.04 million metric tons (DLS-2023-24). Farmers arse focusing on developing comprehensive management and production systems that prioritize higher milk production. Despite these improvements, the most significant obstacle they face is the marketing and distribution of the milk itself. Still, most of the milk is sold in the old way, through informal marketing channels via intermediaries from producers to consumers. Informal milk marketing doesn’t ensure the supply of safe milk to consumers, as the market lacks a robust monitoring system; on the other hand, producers are losing profits due to the fluctuating milk market. The quality of the milk is questionable here, according to the satisfaction level of consumers. Due to the irregular checks of raw milk marketing by the controlling authority, some middlemen use different types of adulterants and preservatives to increase the volume and shelf life of milk. Therefore, adding locally available herbal preservatives for raw milk preservation could be a helpful solution, (Chandra et al., 2016; Maji et al., 2018; Posan et al., 2023). Numerous studies have demonstrated that essential oils, such as eugenol, thymol and carvacrol, derived from Tulsi can inhibit the growth of pathogenic and spoilage microorganisms. These oils can also disrupt microbial cell membranes and interfere with their metabolic processes, which slows down the spoilage of raw milk. In a hot, humid country like Bangladesh, where there is limited infrastructure for milk preservation, Tulsi can be a reliable natural method for preserving raw milk, as most people in the country have long used Tulsi as a medicinal plant.
 
Objectives
 
1. To assess the effect of Tulsi extract on raw milk preservation with different concentrations in raw milk across different season.
2. To determine the appropriate percentage of Tulsi extract for raw milk preservation.
3. To evaluate the potential of herbal preservatives to act as substitutes for chemical preservatives in raw milk.

MATERIALS AND METHODS

Preparation of Tulsi Extract
 
Tulsi (Ocimum sanctum) leaves were taken from the Garden of ANSVM CAMPUS, Babuganj, Barisal, from July 2022 to June 2023. The fresh mature Tulsi leaves were selected for extract preparation according to (Trivedi et al., 2014).  The leaves were washed and shade dried.
 
The method for extracting Tulsi leaves is described below
 
Sorting out Tulsi leaves thoroughly
↓
Wash with potable water thoroughly
↓
Heating at 65oC for 5 minutes in a 1:4 ratio of water
↓
The heat-treated Tulsi was crushed in the juice maker and water was added to obtain a fine paste.
↓
For the extraction of Tulsi juice, it was filtered through a fine, double-layered, clean muslin cloth.
↓
Tulsi juice was kept at refrigerated temperature(7oC) until use.
 
We took a milk sample of 100 ml in each of 4 different test tubes. test tubes. We labeled these tubes with the following treatment groups: aqueous Tulsi extract control (T0), 0.5% (T1), 1.0% (T2) and 1.5% (T3).

RESULTS AND DISCUSSION

We have studied the effects of different concentrations of Tulsi (Ocimum sanctum) extract on the pH levels, acidity percentage and Direct Microscopic Count (DMC) of milk in different seasons and times in the T0 (without Tulsi extract), T1 (with 0.5% Tulsi extract), T2 (with 1% Tulsi extract) and T3 (1.5% Tulsi extract) groups. The pH levels, Percent (%) of acidity and DMC of milk samples of the control group and those treated with Tulsi extract were monitored at 2-hour intervals over 6 hours under three seasonal conditions (summer, rainy and dry seasons). We have analyzed the data by Analysis of Variance (ANOVA) and Duncan’s multiple range test and compared means.
 
Yij= µ + ai + eij
 
Where,
Yij = Observation value of pH, acidity % of milk and DMC.
µ = Population means.
ai  = Effect of Season (i = =3).
eij = The random error of the season.
       
The information in Table 1, 2 and 3 shows how the pH level, percentage (%) of acidity and DMC count changed over time in milk samples with varying amounts of Tulsi extract during seasonal changes. The findings show how Tulsi extract affected the pH level, acidity percentage and DMC count with time and maintain a low microbial count at higher Tulsi extract concentration.

Table 1: Mean pH Levels ± SD in milk samples for control and Tulsi extract-treated groups at different concentrations under different seasons.



​

Table 2: Mean acidity (%) ±SD in milk samples for control and Tulsi extract-treated groups at different concentrations under different seasons.



​

Table 3: Mean log CFU/ml (± Standard deviation) of total DMC count in milk samples with different treatment groups.


 
Summer season
 
In the summer, the pH values and acidity percentage of milk increased across all extract concentrations and control groups, but they were comparatively lower in milk samples with Tulsi extract. Initially, all treatment groups had similar pH levels. Over time, the pH level in the milk samples gradually decreased, consistent with the findings of Marouf and Elmhal (2017), Yang et al., (2023) and Aydogdu et al., (2023). After 2 hours, the control group milk had a pH of (6.32±0.12), while the T1, T2 and T3 groups had a pH of (6.61±0.21,6.62±0.18 and 6.65±0.15). It indicates that with a higher concentration of Tulsi extract, the decrease in pH values, were comparatively lower. The potential antibacterial properties of Tulsi extract may contribute to the observed  pH during summer when it is present. According to Raghav and Saini (2018), Anand et al., (2016), Anbarasu and Vijayalakshmi (2007) and Okafor  et al. (2021), the higher pH in milk treated with Tulsi also implies that Tulsi may act as a buffer to prevent abrupt pH fluctuations, which is essential for preventing spoilage. However, milk samples with higher concentrations of Tulsi, especially at 1% and 1.5%, showed relatively stable pH. It may be due to ursolic acid and eugenol in Tulsi that can influence microbial fermentation and inhibit microbial growth, which may explain the lower acidity in milk (Sharma et al., 2001; Hanaa et al., 2016), thereby affecting pH levels. This suggests that Tulsi extract might help maintain pH stability, which is important for milk preservation. Tulsi leaves are rich in phenolic compounds, which can interact with milk proteins, potentially altering their structure and stability, thus influencing the bioavailability of amino acids (Tosif et al., 2021) and likely also due to the antimicrobial effects of Tulsi in reducing acidification (Khan et al., 2020; Zdolec et al., 2024; Bansavatar et al., 2015; Mehta and Sharma, 2020; Goyal and Chauhan, 2021). The level of significance observed in the statistical analysis at 4 and 6 hours (p<0.05) might be indicative of these findings. A direct microscopic count revealed no significant differences in bacterial growth between the control and Tulsi extract-treated groups at the 2-hour interval. However, significant inhibition of growth was noted in the treatment groups compared to the control after 4 and 6 hours (p<0.05). The decrease in pH and increase in the percentage of acidity in milk levels were noted at 4 hours and 6 hours, indicating natural acidification of milk due to bacterial activity.  This buffering capacity may be attributed to extending the shelf life of milk during warmer months. The direct microscopic count of milk showed that, in the summer season, the acidity percentage was slightly lower in Tulsi extract milk samples than in the control group (0.170±0.012). After the 2-hour mark, the level of acidity in the treated groups (T1, T2 and T3) was (0.155±0.021), (0.152±0.010), (0.148±0.011), whereas it was slightly higher for the control group (0.170±0.012). The acidity of the control groups was (0.192±0.015), (0.142±0.035) and (0.138±0.09) during the four hours, while it was significantly lower in the T2 and T3 groups. This decrease was statistically significant (*p<0.05). The result revealed that Tulsi extract helped maintain the pH balance of milk; this was a similar finding reported by Zou et al., (2017). He admitted that microbial growth and acidity increases were greater and reduce the acidity per cent (%) in untreated milk.
       
At 2, 4 and 6 hours, the acidity percentage of Tulsi-treated samples remained lower (T1: (0.188±0.009), (0.178±0.007), (0.172±0.016) and the acidity percentage of the control group was higher (0.210±0.014). However, % of acidity was comparatively lower in milk samples with higher concentrations of Tulsi extracts, which indicates that plant extract rich in bioactive compounds, such as flavonoids, phenolic acids and alkaloids, exhibit antimicrobial properties by disrupting microbial cell membranes, inhibiting enzymatic functions, or interfering with metabolic pathways, was similar to the finding of Dahanukar et al., (2000). Tulsi (Ocimum sanctum) extract has broad-spectrum antibacterial action against lactic acid bacteria and other spoilage germs, as indicated by the research, which indicates that it notably delays the acidification of raw milk. The main bioactive ingredients in Tulsi, including rosmarinic acid, linalool and eugenol, are responsible for this effect. These substances have been found to break down microbial cell membranes and prevent vital enzymatic function (Bhattacharjee and Dutta, 2019). There was a lower Direct Microscopic Count (DMC) count in summer with higher Tulsi extract added samples T1(4.30±0.07, 5.45±0.12, 6.55±0.16), T2(4.25±0.09, 5.10 ±0.11, 6.05±0.14) and T3(4.20±0.10, 4.95±0.09, 5.85±0.13)  compared to the control group (4.35±0.08, 5.95±0.15, 7.20 ±0.18) which was more profound over time. Khan et al., (2020) stated that the addition of Tulsi extract could inhibit the multiplication    of bacterial population in the raw milk samples was an agreement to our study.
 
Rainy season
 
During the rainy season, the effect of Tulsi extract on milk pH mirrored the summer trend. After two hours, all treatment groups (T1: 6.72±0.19, T2: 6.75±0.19, T3: 6.73±0.21) exhibited a significantly higher pH compared to the Tulsi-treated group (6.38±0.14). Tulsi extract could have maintained a more balanced pH in milk even under humid conditions, as seen by the higher values compared to the control group. After four and six hours, a significant change in pH level was noted among different groups. Comparatively lower pH was noted in the Tulsi-treated group compared to the control group. Tulsi’s effect on pH stability decreased, with a slight variation in the pH levels of the milk samples. This might have occurred because of the lower temperatures and higher moisture content in the air (Zou et al., 2017). In milder seasons, microbial growth is decreased and maintaining a stable pH is essential, especially for preserving milk’s freshness and preventing spoiling. Many studies have revealed that different concentrations of Tulsi extracts have different impacts on their antimicrobial and pH-buffering effects, with higher concentrations sometimes causing different microbial dynamics (Dahanukar et al., 2000). The higher extract levels in the T3 group may have affected the overall composition of the milk, causing slight pH fluctuations.
       
A similar trend for acidity was noted in the rainy season in Tulsi-treated milk samples. During the 2-hour mark, the acidity levels of the control group were 0.150±0.007% and it was slightly lower in the Tulsi-treated groups (T1: 0.148±0.010, T2: 0.142±0.035, T3: 0.138±0.009%), which was statistically significant (*p<0.05). According to Zou et al., (2017), this could be because Tulsi extract keeps milk’s pH stable even in humid environments, delaying its acidification. After 4 hours, the acidity level in the control group was (0.18±0.008)%, whereas the Tulsi-treated groups showed comparatively slower increase of acidity (T1: 0.165±0.008, T2: 0.152±0.009%, T3: 0.149±0.015). At 6 hours, a slow rise in acidity was still evident in the Tulsi-treated samples, though the effect was less pronounced than in the summer season (T1: 0.180±0.007, T2: 0.162±0.010, T3: 0.156±0.011). During the rainy season, the lower microbial activity due to low temperature may help maintain milk pH stability and freshness. It was also evident by the Direct Microscopic Count at 4 and 6 hours (5.70±0.13, 6.90±0.17) for T1, (5.40±0.12, 6.40±0.15) for T2 and (5.25±0.11, 6.20±0.14) hours compared to the control group (6.15±0.14, 7.10±0.19), where there was a significant difference between the control and Tulsi-treated group of samples (P<0.05) was similar to the finding of the acid production of the milk bacteria can decrease by the phytochemicals present in Tulsi extracts consequently increased shelf life up to 8 to 9 h Joshi et al., (2011) and Khan et al., (2020).
 
Dry season
 
Compared to the summer and rainy seasons, the pH data during the dry season showed a less clear trend. All Tulsi treatments showed a slight increase in pH at two hours, with no significant difference between the control and Tulsi-treated groups, where the control had the highest pH of (6.54±0.11). The pH remained stable across different extract concentrations but decreased as time increased to 4 and 6 hours. For example, the control and T3 groups exhibited greater variability over six hours, whereas the T1 and T2 groups maintained more consistent pH values.
       
The decrease in pH at 6 hours in the control group could be a result of natural microbial fermentation, which is still active in the dry season, more slowly compared to the rainy season (Rahman et al., 2015). The antimicrobial properties of Tulsi extract may slow this fermentation process, contributing to a less acidic environment. However, the pH level of higher extract concentrations (T3) of high concentrations of Tulsi may not act linearly and it could introduce slight imbalances in milk composition, affecting pH stability as revealed by (Khan et al., 2020) and (Mittal et al., 2020).
       
The same pattern was noted in the dry season, where milk samples with Tulsi had lower in acidity per cent (%) after the 2-hour mark, with acidity levels in the treated groups (T1: 0.158±0.006, T2: 0.142±0.009, T3: 0.148±0.008) being slightly lower than those in the control (0.158±0.006).
       
The acidity level of the control group was 0.180±0.008 after 4-hour periods, whereas the treated samples had a lower level (T1: 0.168±0.006, T2: 0.152±0.008 and T3: 0.165±0.032%), revealing a statistically significant decrease (*p<0.05). This indicates that Tulsi extract plays a crucial role in slowing down the rate of acidification, which is particularly essential in hotter, drier environments where microbial activity can still be significant. Because microbial load was 4.25±0.07 after two hours in control group, whereas it was 4.22±0.08 in T1, 4.20 ±0.06 for T2 and 4.18±0.09 in T3 group. This was further evidence that the 6-hour milk preservation, which may be explained by an increased amount of Tulsi extract affecting the milk composition by controlling microbial load (7.00±0.17) in control group, (6.40±0.15) in T1, (5.90±0.13) in T2 and (5.70±0.12) in T3 group was supported by Dahanukar et al., (2000).

CONCLUSION

The present study shows that Tulsi extract significantly affects milk’s pH stability, with different impacts across seasons. In summer and rainy seasons, Tulsi extract helped keep a more stable and less acidic pH, which is important for milk preservation. Evidence indicates a lower microbial count with increased Tulsi content across all seasons, with more noticeable effects over time. The dry season exhibited a more complex interaction, with effects that varied based on extract concentration. The level of Tulsi extract was crucial, with higher concentrations generally resulting in more stable pH values, though sometimes causing some variability. As natural preservatives Tulsi may be cost effective where there is a lack of refrigeration and cold chain facilities. Future research should investigate microbial activity and the chemical makeup of milk treated with Tulsi extract to understand the mechanisms behind these effects better.

ACKNOWLEDGEMENT

The author is thankful to the Research and Training Institute of Patuakhali Science and Technology and Thanks to the university for providing the research grant. The author is also grateful to the staff of the Dairy Science Department for their assistance with the entire research.
 
Author contributions
 
Tanni Chanda made important contributions to the study’s conception and design, data acquisition and data analysis and interpretation. Tanni Chanda conducted the experiments and was accountable for aspects of the work and ensured that questions related to the accuracy or integrity of any part of the study would be appropriately investigated and resolved. Tanni Chanda provided final approval of this version of the manuscript for publication. I am involved in drafting the manuscript or revising it critically for important intellectual content. All authors read and approved of the final manuscript.
 
Fund statement
 
Research Tanning Center (RTC) of Patuakhali Science and Technology University.

CONFLICT OF INTEREST

 The authors declare no conflict of interest.

REFERENCES


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    Full Research Article

    Efficacy of Tulsi (Ocimum sanctum) Extract for Preserving Raw Milk in the Informal Marketing System of Bangladesh Across Different Seasons 

    T
    Tanni Chanda1,*
    A
    Arifur Rahman1
    I
    Iftakher Alam Sarkar2
    N
    Naeem Hossain1
    G
    Goutam Kumar Debnath3
    1Department of Dairy Science, Faculty of Animal Science and Veterinary Medicine, Patuakhali Science and Technology University, Barisal 8602, Bangladesh.
    2Buffalo Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341, Bangladesh.
    3Department of Dairy and Poultry Science, Faculty of Veterinary Medicine, Chattogram Veterinary and Animal Sciences University. Chattogram 4225, Bangladesh.

    ABSTRACT

    Background: Like other Southeast Asian countries, Bangladesh’s informal milk market frequently faces issues of contamination due to its high perishability and inadequate cooling facilities. To resolve this issue, this study investigated the use of Tulsi (Ocimum sanctum) leaf extract in different concentrations as a natural and secure preservative.

    Methods: In this study, we included a control group and applied concentrations of 0.5%, 1% and 1.5% aqueous Tulsi leaf extract to raw milk, monitoring their effects over six hours at two-hour intervals across summer, rainy and winter seasons. The sampling was conducted in the Babuganj area of Barisal, Bangladesh, from July 2022 to June 2023. Different parameters, such as pH level, percent (%) of acidity and microbial count of different samples, were measured at the Dairy lab. of the Faculty of Animal Sciences and Veterinary Medicine of Patuakhali Science and Technology University.

    Result: The 1.5% Tulsi extract (T2) was found to be most effective in maintaining pH stability, controlling acidity and microbial load. However, this higher concentration (T3) caused a noticeable color change in the milk. Conversely, the lower 0.5% concentration (T1) demonstrated lower effectiveness in controlling microbial growth (DMC). The 1% concentration (T2) is identified as a better alternative to synthetic preservatives, balancing efficacy with minimal sensory alteration and controlling microbial activity. Further research is needed on its long-term effects.

    INTRODUCTION

    Dairy is an emerging sector in Bangladesh with significant potential to contribute substantially to economic growth. Historically, Bangladesh has been an agricultural country, and the lives of Bangladeshi people are closely related to agriculture. Dairying is a principal part of Agriculture that has an intimate relation to livelihood, especially in rural areas. Over time, the features of dairying have been transformed from a rural livelihood to a highly profitable industry. The number of cattle populations is now 25.01 million, with milk production of 15.04 million metric tons (DLS-2023-24). Farmers arse focusing on developing comprehensive management and production systems that prioritize higher milk production. Despite these improvements, the most significant obstacle they face is the marketing and distribution of the milk itself. Still, most of the milk is sold in the old way, through informal marketing channels via intermediaries from producers to consumers. Informal milk marketing doesn’t ensure the supply of safe milk to consumers, as the market lacks a robust monitoring system; on the other hand, producers are losing profits due to the fluctuating milk market. The quality of the milk is questionable here, according to the satisfaction level of consumers. Due to the irregular checks of raw milk marketing by the controlling authority, some middlemen use different types of adulterants and preservatives to increase the volume and shelf life of milk. Therefore, adding locally available herbal preservatives for raw milk preservation could be a helpful solution, (Chandra et al., 2016; Maji et al., 2018; Posan et al., 2023). Numerous studies have demonstrated that essential oils, such as eugenol, thymol and carvacrol, derived from Tulsi can inhibit the growth of pathogenic and spoilage microorganisms. These oils can also disrupt microbial cell membranes and interfere with their metabolic processes, which slows down the spoilage of raw milk. In a hot, humid country like Bangladesh, where there is limited infrastructure for milk preservation, Tulsi can be a reliable natural method for preserving raw milk, as most people in the country have long used Tulsi as a medicinal plant.
     
    Objectives
     
    1. To assess the effect of Tulsi extract on raw milk preservation with different concentrations in raw milk across different season.
    2. To determine the appropriate percentage of Tulsi extract for raw milk preservation.
    3. To evaluate the potential of herbal preservatives to act as substitutes for chemical preservatives in raw milk.

    MATERIALS AND METHODS

    Preparation of Tulsi Extract
     
    Tulsi (Ocimum sanctum) leaves were taken from the Garden of ANSVM CAMPUS, Babuganj, Barisal, from July 2022 to June 2023. The fresh mature Tulsi leaves were selected for extract preparation according to (Trivedi et al., 2014).  The leaves were washed and shade dried.
     
    The method for extracting Tulsi leaves is described below
     
    Sorting out Tulsi leaves thoroughly
    ↓
    Wash with potable water thoroughly
    ↓
    Heating at 65oC for 5 minutes in a 1:4 ratio of water
    ↓
    The heat-treated Tulsi was crushed in the juice maker and water was added to obtain a fine paste.
    ↓
    For the extraction of Tulsi juice, it was filtered through a fine, double-layered, clean muslin cloth.
    ↓
    Tulsi juice was kept at refrigerated temperature(7oC) until use.
     
    We took a milk sample of 100 ml in each of 4 different test tubes. test tubes. We labeled these tubes with the following treatment groups: aqueous Tulsi extract control (T0), 0.5% (T1), 1.0% (T2) and 1.5% (T3).

    RESULTS AND DISCUSSION

    We have studied the effects of different concentrations of Tulsi (Ocimum sanctum) extract on the pH levels, acidity percentage and Direct Microscopic Count (DMC) of milk in different seasons and times in the T0 (without Tulsi extract), T1 (with 0.5% Tulsi extract), T2 (with 1% Tulsi extract) and T3 (1.5% Tulsi extract) groups. The pH levels, Percent (%) of acidity and DMC of milk samples of the control group and those treated with Tulsi extract were monitored at 2-hour intervals over 6 hours under three seasonal conditions (summer, rainy and dry seasons). We have analyzed the data by Analysis of Variance (ANOVA) and Duncan’s multiple range test and compared means.
     
    Yij= µ + ai + eij
     
    Where,
    Yij = Observation value of pH, acidity % of milk and DMC.
    µ = Population means.
    ai  = Effect of Season (i = =3).
    eij = The random error of the season.
           
    The information in Table 1, 2 and 3 shows how the pH level, percentage (%) of acidity and DMC count changed over time in milk samples with varying amounts of Tulsi extract during seasonal changes. The findings show how Tulsi extract affected the pH level, acidity percentage and DMC count with time and maintain a low microbial count at higher Tulsi extract concentration.

    Table 1: Mean pH Levels ± SD in milk samples for control and Tulsi extract-treated groups at different concentrations under different seasons.



    ​

    Table 2: Mean acidity (%) ±SD in milk samples for control and Tulsi extract-treated groups at different concentrations under different seasons.



    ​

    Table 3: Mean log CFU/ml (± Standard deviation) of total DMC count in milk samples with different treatment groups.


     
    Summer season
     
    In the summer, the pH values and acidity percentage of milk increased across all extract concentrations and control groups, but they were comparatively lower in milk samples with Tulsi extract. Initially, all treatment groups had similar pH levels. Over time, the pH level in the milk samples gradually decreased, consistent with the findings of Marouf and Elmhal (2017), Yang et al., (2023) and Aydogdu et al., (2023). After 2 hours, the control group milk had a pH of (6.32±0.12), while the T1, T2 and T3 groups had a pH of (6.61±0.21,6.62±0.18 and 6.65±0.15). It indicates that with a higher concentration of Tulsi extract, the decrease in pH values, were comparatively lower. The potential antibacterial properties of Tulsi extract may contribute to the observed  pH during summer when it is present. According to Raghav and Saini (2018), Anand et al., (2016), Anbarasu and Vijayalakshmi (2007) and Okafor  et al. (2021), the higher pH in milk treated with Tulsi also implies that Tulsi may act as a buffer to prevent abrupt pH fluctuations, which is essential for preventing spoilage. However, milk samples with higher concentrations of Tulsi, especially at 1% and 1.5%, showed relatively stable pH. It may be due to ursolic acid and eugenol in Tulsi that can influence microbial fermentation and inhibit microbial growth, which may explain the lower acidity in milk (Sharma et al., 2001; Hanaa et al., 2016), thereby affecting pH levels. This suggests that Tulsi extract might help maintain pH stability, which is important for milk preservation. Tulsi leaves are rich in phenolic compounds, which can interact with milk proteins, potentially altering their structure and stability, thus influencing the bioavailability of amino acids (Tosif et al., 2021) and likely also due to the antimicrobial effects of Tulsi in reducing acidification (Khan et al., 2020; Zdolec et al., 2024; Bansavatar et al., 2015; Mehta and Sharma, 2020; Goyal and Chauhan, 2021). The level of significance observed in the statistical analysis at 4 and 6 hours (p<0.05) might be indicative of these findings. A direct microscopic count revealed no significant differences in bacterial growth between the control and Tulsi extract-treated groups at the 2-hour interval. However, significant inhibition of growth was noted in the treatment groups compared to the control after 4 and 6 hours (p<0.05). The decrease in pH and increase in the percentage of acidity in milk levels were noted at 4 hours and 6 hours, indicating natural acidification of milk due to bacterial activity.  This buffering capacity may be attributed to extending the shelf life of milk during warmer months. The direct microscopic count of milk showed that, in the summer season, the acidity percentage was slightly lower in Tulsi extract milk samples than in the control group (0.170±0.012). After the 2-hour mark, the level of acidity in the treated groups (T1, T2 and T3) was (0.155±0.021), (0.152±0.010), (0.148±0.011), whereas it was slightly higher for the control group (0.170±0.012). The acidity of the control groups was (0.192±0.015), (0.142±0.035) and (0.138±0.09) during the four hours, while it was significantly lower in the T2 and T3 groups. This decrease was statistically significant (*p<0.05). The result revealed that Tulsi extract helped maintain the pH balance of milk; this was a similar finding reported by Zou et al., (2017). He admitted that microbial growth and acidity increases were greater and reduce the acidity per cent (%) in untreated milk.
           
    At 2, 4 and 6 hours, the acidity percentage of Tulsi-treated samples remained lower (T1: (0.188±0.009), (0.178±0.007), (0.172±0.016) and the acidity percentage of the control group was higher (0.210±0.014). However, % of acidity was comparatively lower in milk samples with higher concentrations of Tulsi extracts, which indicates that plant extract rich in bioactive compounds, such as flavonoids, phenolic acids and alkaloids, exhibit antimicrobial properties by disrupting microbial cell membranes, inhibiting enzymatic functions, or interfering with metabolic pathways, was similar to the finding of Dahanukar et al., (2000). Tulsi (Ocimum sanctum) extract has broad-spectrum antibacterial action against lactic acid bacteria and other spoilage germs, as indicated by the research, which indicates that it notably delays the acidification of raw milk. The main bioactive ingredients in Tulsi, including rosmarinic acid, linalool and eugenol, are responsible for this effect. These substances have been found to break down microbial cell membranes and prevent vital enzymatic function (Bhattacharjee and Dutta, 2019). There was a lower Direct Microscopic Count (DMC) count in summer with higher Tulsi extract added samples T1(4.30±0.07, 5.45±0.12, 6.55±0.16), T2(4.25±0.09, 5.10 ±0.11, 6.05±0.14) and T3(4.20±0.10, 4.95±0.09, 5.85±0.13)  compared to the control group (4.35±0.08, 5.95±0.15, 7.20 ±0.18) which was more profound over time. Khan et al., (2020) stated that the addition of Tulsi extract could inhibit the multiplication    of bacterial population in the raw milk samples was an agreement to our study.
     
    Rainy season
     
    During the rainy season, the effect of Tulsi extract on milk pH mirrored the summer trend. After two hours, all treatment groups (T1: 6.72±0.19, T2: 6.75±0.19, T3: 6.73±0.21) exhibited a significantly higher pH compared to the Tulsi-treated group (6.38±0.14). Tulsi extract could have maintained a more balanced pH in milk even under humid conditions, as seen by the higher values compared to the control group. After four and six hours, a significant change in pH level was noted among different groups. Comparatively lower pH was noted in the Tulsi-treated group compared to the control group. Tulsi’s effect on pH stability decreased, with a slight variation in the pH levels of the milk samples. This might have occurred because of the lower temperatures and higher moisture content in the air (Zou et al., 2017). In milder seasons, microbial growth is decreased and maintaining a stable pH is essential, especially for preserving milk’s freshness and preventing spoiling. Many studies have revealed that different concentrations of Tulsi extracts have different impacts on their antimicrobial and pH-buffering effects, with higher concentrations sometimes causing different microbial dynamics (Dahanukar et al., 2000). The higher extract levels in the T3 group may have affected the overall composition of the milk, causing slight pH fluctuations.
           
    A similar trend for acidity was noted in the rainy season in Tulsi-treated milk samples. During the 2-hour mark, the acidity levels of the control group were 0.150±0.007% and it was slightly lower in the Tulsi-treated groups (T1: 0.148±0.010, T2: 0.142±0.035, T3: 0.138±0.009%), which was statistically significant (*p<0.05). According to Zou et al., (2017), this could be because Tulsi extract keeps milk’s pH stable even in humid environments, delaying its acidification. After 4 hours, the acidity level in the control group was (0.18±0.008)%, whereas the Tulsi-treated groups showed comparatively slower increase of acidity (T1: 0.165±0.008, T2: 0.152±0.009%, T3: 0.149±0.015). At 6 hours, a slow rise in acidity was still evident in the Tulsi-treated samples, though the effect was less pronounced than in the summer season (T1: 0.180±0.007, T2: 0.162±0.010, T3: 0.156±0.011). During the rainy season, the lower microbial activity due to low temperature may help maintain milk pH stability and freshness. It was also evident by the Direct Microscopic Count at 4 and 6 hours (5.70±0.13, 6.90±0.17) for T1, (5.40±0.12, 6.40±0.15) for T2 and (5.25±0.11, 6.20±0.14) hours compared to the control group (6.15±0.14, 7.10±0.19), where there was a significant difference between the control and Tulsi-treated group of samples (P<0.05) was similar to the finding of the acid production of the milk bacteria can decrease by the phytochemicals present in Tulsi extracts consequently increased shelf life up to 8 to 9 h Joshi et al., (2011) and Khan et al., (2020).
     
    Dry season
     
    Compared to the summer and rainy seasons, the pH data during the dry season showed a less clear trend. All Tulsi treatments showed a slight increase in pH at two hours, with no significant difference between the control and Tulsi-treated groups, where the control had the highest pH of (6.54±0.11). The pH remained stable across different extract concentrations but decreased as time increased to 4 and 6 hours. For example, the control and T3 groups exhibited greater variability over six hours, whereas the T1 and T2 groups maintained more consistent pH values.
           
    The decrease in pH at 6 hours in the control group could be a result of natural microbial fermentation, which is still active in the dry season, more slowly compared to the rainy season (Rahman et al., 2015). The antimicrobial properties of Tulsi extract may slow this fermentation process, contributing to a less acidic environment. However, the pH level of higher extract concentrations (T3) of high concentrations of Tulsi may not act linearly and it could introduce slight imbalances in milk composition, affecting pH stability as revealed by (Khan et al., 2020) and (Mittal et al., 2020).
           
    The same pattern was noted in the dry season, where milk samples with Tulsi had lower in acidity per cent (%) after the 2-hour mark, with acidity levels in the treated groups (T1: 0.158±0.006, T2: 0.142±0.009, T3: 0.148±0.008) being slightly lower than those in the control (0.158±0.006).
           
    The acidity level of the control group was 0.180±0.008 after 4-hour periods, whereas the treated samples had a lower level (T1: 0.168±0.006, T2: 0.152±0.008 and T3: 0.165±0.032%), revealing a statistically significant decrease (*p<0.05). This indicates that Tulsi extract plays a crucial role in slowing down the rate of acidification, which is particularly essential in hotter, drier environments where microbial activity can still be significant. Because microbial load was 4.25±0.07 after two hours in control group, whereas it was 4.22±0.08 in T1, 4.20 ±0.06 for T2 and 4.18±0.09 in T3 group. This was further evidence that the 6-hour milk preservation, which may be explained by an increased amount of Tulsi extract affecting the milk composition by controlling microbial load (7.00±0.17) in control group, (6.40±0.15) in T1, (5.90±0.13) in T2 and (5.70±0.12) in T3 group was supported by Dahanukar et al., (2000).

    CONCLUSION

    The present study shows that Tulsi extract significantly affects milk’s pH stability, with different impacts across seasons. In summer and rainy seasons, Tulsi extract helped keep a more stable and less acidic pH, which is important for milk preservation. Evidence indicates a lower microbial count with increased Tulsi content across all seasons, with more noticeable effects over time. The dry season exhibited a more complex interaction, with effects that varied based on extract concentration. The level of Tulsi extract was crucial, with higher concentrations generally resulting in more stable pH values, though sometimes causing some variability. As natural preservatives Tulsi may be cost effective where there is a lack of refrigeration and cold chain facilities. Future research should investigate microbial activity and the chemical makeup of milk treated with Tulsi extract to understand the mechanisms behind these effects better.

    ACKNOWLEDGEMENT

    The author is thankful to the Research and Training Institute of Patuakhali Science and Technology and Thanks to the university for providing the research grant. The author is also grateful to the staff of the Dairy Science Department for their assistance with the entire research.
     
    Author contributions
     
    Tanni Chanda made important contributions to the study’s conception and design, data acquisition and data analysis and interpretation. Tanni Chanda conducted the experiments and was accountable for aspects of the work and ensured that questions related to the accuracy or integrity of any part of the study would be appropriately investigated and resolved. Tanni Chanda provided final approval of this version of the manuscript for publication. I am involved in drafting the manuscript or revising it critically for important intellectual content. All authors read and approved of the final manuscript.
     
    Fund statement
     
    Research Tanning Center (RTC) of Patuakhali Science and Technology University.

    CONFLICT OF INTEREST

     The authors declare no conflict of interest.

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