Key findings:

The study found that adding butterfly pea flower extract to pasteurized milk significantly affected the total bacterial count and pH over 20 days at low temperatures, following a cubic regression pattern. However, the extract did not significantly inhibit E. coli, showing no consistent relationship.

What is known and what is new?

It is known that fresh milk has a short shelf life and that pasteurization extends this shelf life by reducing bacterial content. This study introduces a new finding by exploring the effect of adding butterfly pea flower extract to pasteurized milk. It was discovered that while the extract significantly influenced the total bacterial count and pH over 20 days of low-temperature storage, it did not significantly inhibit E. coli, presenting a novel insight into the antibacterial properties of butterfly pea flower extract in pasteurized milk.

What is the implication, and what should change now?

The implication of this study is that adding butterfly pea flower extract to pasteurized milk can effectively influence the total bacterial count and pH during storage, potentially enhancing the milk's shelf life and stability. However, since it does not significantly inhibit E. coli, producers should not rely on it solely for antibacterial purposes. Dairy producers may consider incorporating butterfly pea flower extract to improve shelf life but should continue to use other methods for effective antibacterial control.

Indonesian people are starting to realize the importance of consuming milk, which is one of the livestock products during the Covid-19 pandemic. This can be seen from data from the Central Statistics Agency (2021) which shows that there has been an increase of 0.25 percent from 2019, where the level of milk consumption per capita of the Indonesian people in 2020 is 16.27 kg/capita/year. The level of milk consumption per capita in Indonesia is expected to continue to increase from year to year. Pasteurization is one of the easiest ways to do at home in preventing damage to milk and can maintain the appearance and taste of fresh milk to a minimum without losing the nutrients it contains. However, pasteurized milk has a shelf life that generally ranges from 5-7 days.

One of the factors that can affect the shelf life of milk is contamination by microorganism. It is caused milk is the best medium for bacterial growth. Although milk is stored at refrigeration temperature, it will experience an increase in colony count, although not significant. However, this indicates that microbes can still grow during storage at low temperatures. The type and number of bacteria can be influenced by several things, such as the microbial population of fresh milk and other raw materials such as sugar, the perfection of the pasteurization process, packaging the speed of storage in the cooler [1].

One of the bacteria that can contaminate milk is Escherichia coli. E. coli is a gram-negative bacterium found in the human body, but if the amount exceeds the normal limit, it is classified as a pathogenic bacterium that can cause disease. If E. coli in large quantities enters the body of someone whose immune system is weakened, it can endanger people health and it will cause diseases such as diarrhea, fever, stomach cramps, and vomiting.

In addition, storage also affects the acidity intensity (pH) value of pasteurized milk [2]. This is because the pH value of milk can be influenced by the amount of lactic acid, which is the result of the decomposition of lactose by bacteria and enzyme activity contained in milk. If the number of bacteria in pasteurized milk increases, the production of lactic acid will also increase, resulting in a lower pH value in the milk. 

Butterfly pea flower extract has active compounds, namely flavonoids, tannins, saponins, terpenoids, and alkaloids [3]. The active content has the potential as an antibacterial which is known to inhibit the growth of several types of bacteria, one of which is E. coli [4]. It aims to reduce the number of colonies of contaminant bacteria contained in pasteurized milk by adding butterfly pea flower extract to pasteurized milk. The consumers who consume this drink avoid diseases caused by pathogenic bacteria, especially E. coli. Based on this description, the authors are interested in further research on the effect of the addition of butterfly pea flower extract to pasteurized milk on the total number of bacteria, inhibition (Escherichia coli), and pH during low temperature storage (4-6 ^oC).

Experimental section

The materials used are fresh cow's milk, butterfly pea flowers, 50 ml plastic bottles, distilled water, antibiotic chloramphenicol, disposable Petri dishes, cotton swabs, nutrient agar (NA) media, physiological NaCl, 70% alcohol, and methylated spirit. The method used in this research is an experimental method using a complete randomized design (RAL). There were 4 treatments in this study, namely P₁ (5th-day storage), P₂ (10th-day storage), P₃ (15th-day storage), and P₄ (20th-day storage). The results of this study were analyzed with the orthogonal polynomial mathematical model as follows according to [5]:

Description:

α             = interception parameter

           = partial regression coefficient associated with the i-th polynomial degree (i = 1, 2, …, n)

Y              = response variable being studied or observed

X              = treatment of storage duration at low temperature

ϵ              = error which is a stochastic random variable

Assumptions:

1. Influence is fixed.

2. Normal distribution of data with mean = 0 and variance of .

Preparation of Butterfly Pea Flower Pasteurized Milk

Fresh butterfly pea flowers that have been washed and dried using an oven at 40^oC for 22 hours. Dried butterfly pea flowers were crushed with a blender until smooth and sieved with a sieve to get the powder. The powder was dissolved using distilled water (1:5) and heated at 62^oC for 30 minutes [6]. Mix 10% butterfly pea flower extract in milk that has been pasteurized at 72^oC for 15 seconds. Perform sample storage using a container in the form of a 100 ml plastic bottle which is given a plastic wrap on the bottle cap and placed in a refrigerator at a temperature of 4-6^oC for 20 days.

Calculation of Total Bacterial Count

Media preparation is done by dissolving NA media in distilled water and followed by sterilization in an autoclave at 121^oC for 15 minutes. A homogeneous sample of 1 ml was poured with a sterile pipette into test tube I containing physiological NaCl (10^-1) and shaken with a vortex. Then 1 mL of test tube I suspension was transferred to test tube II, and so on up to the desired dilution. Put 1 ml of sample and 25 ml of NA into a sterile petri dish, then shake the petri dish in a clockwise or figure eight motion 5 times so that the sample solution and NA media are well mixed. Position the plate horizontally and wait for it to harden. After that, position the plate with the lid down and put it in a 37°C incubator for 24 hours. The number of colonies after 24 hours (25-250 colonies) was counted and the calculation results were interpreted based on the following formula [7]:

N =

Description:

N             = Number of colonies

C              = Total number of colonies on the counted plate

n₁            = Number of colonies in the first dilution

n₂            = Number of colonies in the second dilution

d              = The first dilution counted

Inhibition Test against E. coli Antibacterial

Media preparation is done by dissolving NA media in distilled water, followed by sterilization in an autoclave at 121^oC for 15 minutes. 25 ml of media was poured into a plate and allowed to solidify. E. coli test colonies were suspended by taking one inoculating loop of colonies from solid NA media into a test tube containing 5 ml of physiological NaCl and standardizing the turbidity of the colony suspension with 0.5 McFarland standard (approximately 1.5 x 10⁸ CFU/ml). The test bacterial suspension was inoculated on 0.1 ml of NA media concentration and leveled with a hockey stick. After drying, wells were made using the tip of a sterile pipette, and 75 µL of the sample was inserted into them. Incubate for 24 hours at 37^oC and the resulting inhibition zone is measured using a caliper three times in different positions and the average value is calculated [8].

Measurement of pH Value

Calibration of the pH meter is done by dipping the electrode tip into a buffer solution (pH 4 and 7 as controls). After that, the electrode tip of the pH meter is dipped in a sample of 10 ml and waited until it showed a stable number [9].

The observation data of the storage duration of pasteurized milk with the addition of butterfly pea flower extract at low temperature (4 - 6^oC) for 20 days are presented in Table 1.

Table 1. Observation data of the storage duration of pasteurized milk with the addition of butterfly pea flower extract at low temperature (4 - 6^oC) for 20 days.

Description:

P₁      : 1st treatment (5th day of storage)

P₂      : 2nd treatment (10th day of storage)

P₃      : 3rd treatment (15th day of storage)

P₄      : 4th treatment (20th day of storage)

Effect of Storage Duration at Low Temperature (4-6^oC) on Total Bacterial Count of Butterfly Pea Flower Milk

Figure 1. Graph of the total bacterial count of pasteurized milk with the addition of butterfly pea flower extract during low temperature storage (4-6^oC)

The results of the observation of the storage time of pasteurized milk with the addition of butterfly pea flower extract for 20 days resulted in an average value of total bacterial counts that varied in each treatment, namely in the range of 2.70 x 10⁴ - 3.89 x 10⁴ cfu/ml. This shows that the longer the storage at low temperature (4-6^oC) for 20 days, the more the total number of bacteria contained in pasteurized milk with the addition of butterfly pea flower extract will increase. Based on the analysis of variance, it can be seen that the storage treatment at low temperature (4-6^oC) is significantly different (P <0.05) from the total bacterial count of pasteurized milk with the addition of butterfly pea flower extract. Figure 1

Based on the graph of orthogonal polynomial test results (Figure 1.) shows a significant difference in cubic regression with the equation y = – 0.0004x³ + 0.0169x² – 0.1955x + 5.0374 with a high level of determination of 0.973. The Length of storage at low temperature until day 20 had an effect of 97.3% on the total bacterial count of pasteurized milk with the addition of butterfly pea flower extract. The graph of the total bacterial count of pasteurized milk with the addition of butterfly pea flower extract produced resembles the growth curve of bacteria in general. In line with the opinion of Wang et al (2015) [10] which states that the bacterial growth curve is divided into several phases, namely the lag phase, log phase, stationary phase, and death phase. 

Butterfly pea flower extract is thought to also influence the total number of bacteria contained in the sample. The antimicrobial activity works on the sample due to the content of phenol compounds, tannins, saponins, alkaloids, and flavonoids contained in the extract. This is supported by the opinion of Al-Snafi et al (2016) [11] which states that butterfly pea flower extract shows its inhibitory power as an antibacterial substance against several bacteria, such as Klebsiella pneumonia, Bacillus subtilis, Pseudomonas aeruginosa, Streptococcus agalactiae, Escherichia coli, Aeromonas hydrophila, and Aeromonas formicans.

The total number of bacteria contained in pasteurized milk with the addition of butterfly pea flower extract is still considered safe for consumption after experiencing storage for 20 days. According to SNI 8984 (2021) [12], the limit of the total number of bacteria contained in plain liquid milk is 5 x 10⁵ colonies/ml, which means that this dairy product is still safe and can be consumed. If the total number of bacteria contained in pasteurized milk exceeds this standard and is consumed by humans, it will have an impact on health.

Effect of Length of Storage at Low Temperature (4 - 6^oC) on Inhibition (E. coli) of Butterfly Pea Flower Milk

The observation of inhibition against antibacterial E. coli of pasteurized milk with the addition of butterfly pea flower extract with storage at low temperature (4 - 6^oC) for 20 days has a range of 5.05 - 7.10 mm. Based on the analysis of variance, it can be seen that the storage treatment at low temperature (4-6^oC) is not significantly different (P ≥ 0.05) on the antibacterial inhibition of E. coli of pasteurized milk with the addition of butterfly pea flower extract. Furthermore, the orthogonal polynomial test was conducted to determine the equation between the response and treatment. The result is not significantly different in each regression so it does not have the best equation.

Based on the observation, it can be seen that the average value of antibacterial inhibition against E. coli produced by pasteurized milk with the addition of butterfly pea flower is included in the medium category (6 - 10 mm). It is suspected that the extract of butterfly pea flower has antibacterial properties to produce an inhibition zone against E. coli bacteria. Following the opinion of Al-Snafi (2016) [11] which states that the addition of butterfly pea flower extract shows its inhibitory power as an antibacterial substance against several bacteria, one of which is Escherichia coli. The inhibition of the biological activity of various types of bacteria is caused by the presence of phenol compounds, tannins, saponins, alkaloids, and flavonoids in t butterfly pea flower extract.

Gupte (1990) [13] states that the magnitude of the inhibition zone formed around the disk indicates the degree of sensitivity of the bacteria to the antibiotics used. The antibiotic used in this study as a positive control, which has an average value of 18.66 mm is included in the strong category. Pasteurized milk with the addition of butterfly pea flowers has a smaller inhibition (E. coli) than the inhibition of antibiotics as a positive control. Jawetz, et al. (2017) [14] stated that E. coli is a type of gram-negative bacteria that has a cell wall consisting of 3 polymers, namely the outer layer of lipoproteins, the middle layer of lipopolysaccharides, and the inner layer of peptidoglycan. So some types of compounds can damage cell wall tissue.

The antibacterial inhibition test value of E. coli in pasteurized milk added with butterfly pea flower extract had no significant effect at low temperature storage for 20 days. It is suspected that the different extraction methods used will affect the levels of anthocyanins produced. The results of research by Purwaniati et al (2020) [15] showed that the total anthocyanin content obtained in fresh butterfly pea flowers was more than in other forms, namely dry and in tea bag packaging. The choice of temperature during the extraction process also affects the anthocyanin content obtained. The higher the extraction temperature, the more anthocyanin levels are obtained when brewing. However, it should be noted that anthocyanin levels will decrease at a temperature of 100^oC because of the degradation of anthocyanins. 

Effect of Storage Duration at Low Temperature (4 - 6^oC) on the pH Value of Butterfly Pea Flower Milk

The observation of the pH value of pasteurized milk with the addition of butterfly pea flower extract with 4 storage treatments at low temperature (4 - 6^oC) resulted in an average pH value in the range of 6.26 - 6.48. The response of the effect of storage treatment at low temperature (4-6^oC) on the pH value of pasteurized milk with the addition of butterfly pea flower extract can be known from the analysis of variance. The results showed that the treatment of storage at low temperatures (4 - 6^oC) was significantly different (P <0.05) from the pH value of pasteurized milk with the addition of t butterfly pea flower extract. Figure 2

Figure 3. Graph of pH value of pasteurized milk with the addition of butterfly pea flower extract during low temperature storage (4-6^oC)

Furthermore, the orthogonal polynomial test was conducted to determine the regression pattern of pasteurized milk storage treatment with the addition of butterfly pea flower extract to the pH value. The results showed that it was significantly different in cubic regression. The cubic equation is the best estimator equation, namely y = – 0.001x³ + 0.0348x² – 0.3725x + 7.456 with a determination rate of 0.798. Duration of storage at low temperature until day 20 had an effect of 79.8% on the total pH value of pasteurized milk with the addition of butterfly pea flower extract.

This is in line with the research of Maryana et al (2016) [16] which states that pasteurized milk has a pH range of 6.22-6.44. This is thought to be due to butterfly pea extract having an acidic pH value of 5.01. So pasteurized milk with the addition of butterfly pea flower extract has a pH value that is lower than the pH of fresh cow's milk. 

Storage of pasteurized milk with the addition of butterfly pea flower extract for more than 20 days is expected to decrease the pH value, which means that the acidity level of the milk will increase. The acidity level is known to affect the number of microorganisms, where the higher the pH, the higher the number of microorganisms that grow which damages the quality of milk. This can occur due to the activity of psychrophilic bacteria that can convert lactose into amino acids. According to Abrar (2013) [17], psychrophilic bacterial species can damage milk during storage at low temperatures in the refrigerator such as Pseudomonas sp and Proteus sp which can reduce milk acidity. So that the storage of milk in the refrigerator if the storage length is not limited will result in a decrease in the quality of the milk.

Storage of pasteurized milk with the addition of butterfly pea flower extract at low temperature (4-6^oC) for 20 days had a significant effect on the total bacterial count by following a cubic regression pattern in the equation y = – 0.0004x³ + 0.0169x² – 0.1955x + 5.0374 and pH value which had a significant effect by following a cubic regression pattern in the equation y = – 0.001x³ + 0.0348x² – 0.3725x + 7.456, but had no significant effect on the antibacterial inhibition of E. coli and had no relationship pattern.

Conflict of Interest:

The authors declare that they have no conflict of interest.

Funding: No funding sources 

Ethical approval: The study was approved by the Institutional Ethics Committee of Universitas Negeri Makassar

1. Wulandari, Z., E. Taufik, and M. Syarif. "Kajian kualitas produk susu pasteurisasi hasil penerapan rantai pendingin." Jurnal Ilmu Produksi dan Teknologi Hasil Peternakan 5.3 (2017): 94-100. DOI: https://doi.org/10.29244/jipthp.5.3.94-100

2. Habibah, Habibah. "PENGARUH LAMA PASTEURISASI DAN LAMA PENYIMPANAN TERHADAP KUALITAS AIR SUSU SAPI PERAH FRIESIAN HOLSTEIN." BIOSCIENTIAE 8.1 (2018). DOI: https://doi.org/10.20527/b.v8i1.184

3. Wang, Qinghu, et al. "Anti-inflammatory effects, nuclear magnetic resonance identification, and high-performance liquid chromatography isolation of the total flavonoids from Artemisia frigida." Journal of food and drug analysis 24.2 (2016): 385-391. https://doi.org/10.1016/j.jfda.2015.11.004

4. Muhammad Ezzudin, R., and M. S. Rabeta. "A potential of telang tree (Clitoria ternatea) in human health." Food Research 2.5 (2018): 415-420. http://www.myfoodresearch.com/uploads/8/4/8/5/84855864/_2__fr-2018-073_ezzudin.pdf

5. Gaspersz, Vincent. "Teknik analisis dalam penelitian percobaan I." (1995).

6. Nadia, Lana Santika, Adi Sutakwa, and Suharman Suharman. "Pengaruh penambahan ekstrak bunga telang (Clitoria ternatea) terhadap pertumbuhan bakteri asam laktat pada pembuatan yogurt telang." Journal of Food and Culinary (2020): 10-17. DOI: https://doi.org/10.12928/jfc.v3i1.3123

7. Maturin, Larry, and James T. Peeler. "Bacteriological analytical manual." US Food and Drug Administration (2001). 

8. Nurhayati, Lilih Siti, Nadhira Yahdiyani, and Akhmad Hidayatulloh. "Perbandingan pengujian aktivitas antibakteri starter yogurt dengan metode difusi sumuran dan metode difusi cakram." Jurnal Teknologi Hasil Peternakan 1.2 (2020): 41-46.

9. Legowo, Anang Mohamad, and S. Mulyani Kusrahayu. "Ilmu dan Teknologi Susu." Badan Penerbit Universitas Diponegoro, Semarang (2009).

10. Wang, Liyun, et al. "Bacterial growth, detachment and cell size control on polyethylene terephthalate surfaces." Scientific reports 5.1 (2015): 15159. https://www.nature.com/articles/srep15159

11. Al-Snafi, Ali Esmail. "Medicinal plants with antimicrobial activities (part 2): Plant based review." Sch Acad J Pharm 5.6 (2016): 208-239. https://saspublishers.com/media/articles/SAJP-56208-239.pdf

12. Badan Standarisasi Nasional. (2021). SNI 8984:2021 Susu Cair Plain. Jakarta: BSN.

13. Gupte, Satish. "Mikrobiologi dasar." Jakarta: Binarupa Aksara (1990).

14. Jawetz., Melnick., & Adelberg. (2017). Mikrobiologi Kedokteran. Edisi 27. Jakarta: EGC. 

15. Purwaniati, Purwaniati, Ahmad Rijalul Arif, and Anne Yuliantini. "Analisis kadar antosianin total pada sediaan bunga telang (clitoria ternatea) dengan metode pH diferensial menggunakan spektrofotometri visible." Jurnal Farmagazine 7.1 (2020): 18-23. https://www.neliti.com/publications/328909/analisis-kadar-antosianin-total-pada-sediaan-bunga-telang-clitoria-ternatea-deng

16. Maryana, Dwi, Ratmawati Malaka, and Fatma Maruddin. "Karakteristik fisiko-kimia dan organoleptik susu pasteurisasi dengan penambahan ekstrak daun binahong (Anredera Cordifolia (Ten) Steenis) dan Sukrosa." Jurnal Sains dan Teknologi 16.2 (2016): 107-112. 

17. Abrar, Mahdi. "Pengembangan model untuk memprediksi pengaruh suhu penyimpanan terhadap laju pertumbuhan bakteri pada susu segar." Jurnal Medika Veterinaria 7.2 (2013). https://jurnal.usk.ac.id/JMV/article/view/2945