Key findings:

This study on kefir demonstrated that adding date juice significantly impacts pH, total lactic acid bacteria, and yeast levels. The optimal concentration was found to be 30%, yielding a pH of 3.72, 2.94 x 10^7 CFU/mL of lactic acid bacteria, and 1.16 x 10^7 CFU/mL of yeast.

What is known and what is new?

It is known that kefir, a probiotic drink, is made from fermented milk using kefir grains that contain lactic acid bacteria and yeast. This study introduces a new finding by examining the effect of adding date juice at various concentrations on kefir. It was discovered that a 30% concentration of date juice optimally enhances kefir's pH, and increases the total lactic acid bacteria and yeast levels, providing a detailed understanding of how date juice can be used to improve kefir's probiotic properties.

What is the implication, and what should change now?

The implication of this study is that incorporating date juice into kefir production can enhance its probiotic qualities by optimizing the levels of lactic acid bacteria and yeast. As a result, kefir producers should consider adding date juice at a concentration of 30% to improve the health benefits and quality of their products. This finding could lead to a change in kefir manufacturing practices, promoting the use of natural fruit juices to enhance probiotic drinks.

Cow's milk is a type of milk that is widely consumed by the public. The milk is in the form of a yellowish-white liquid obtained from the secretion of the udder glands of lactating cows through the milking process. Every year the need for milk tends to increase, in 2019 the level of milk consumption of the Indonesian people ranged from 16.23 kg/capita/year and increased by 0.25% in 2020 [1]. The increase in food needs is closely related to population growth, nutritional awareness, and the need to improve the quality of life in the community. Increasing public awareness to consume healthier and quality products is a great opportunity that can be done through the development of dairy products in Indonesia. Milk processing is needed to increase added value so that it has more attractiveness for consumption.

The condition of COVID-19 pandemic has made people not just consume drinks and food to meet physical needs, but more importantly, to achieve optimal health and fitness levels. Based on this, people will prefer the food they consume to provide health benefits outside of the main function of the foodstuff. Milk is one of the drinks that has a good impact on the health of the body because it is rich in high-quality nutrients. Unfortunately, milk has the property of being easily damaged so it needs handling and processing, one of which is the fermentation process.

One of the fermented milk products that has a good impact on the health of the body is kefir. Kefir itself is a milk drink that is processed by fermentation using kefir grains as a starter which contains lactic acid bacteria and yeast. During the fermentation process, lactic acid bacteria found in kefir grain will produce lactic acid, while yeast will produce CO₂ and alcohol so that the kefir produced will have a slightly sour taste, the taste of alcohol and soda derived from microbes that work during the fermentation process [2]. The potential of kefir as a functional food will be more developed when combined with natural foods such as fruit, in this case, one of them with the addition of sukari date juice.

Dates (Phoenix dactylifera L.) are fruits that have high nutritional content, especially containing antioxidants, fructose, glucose, dietary fiber, and vitamins. The total sugar content in sukari dates reaches 70.68% with a fiber content of 9.11% [3]. The content of sugar and fiber contained in dates can act as prebiotics, which are natural compounds that become food or energy intake for the growth of bacteria or probiotics in making kefir. The fermentation process plays a very important role as a determinant of the success of lactic acid bacteria in remodeling lactose contained in milk so that the availability of energy obtained from dates is targeted to increase the growth of lactic acid bacteria accompanied by a decrease in pH value and total yeast optimal in kefir products.

The potential development of date juice kefir products can increase the functional effectiveness of the date fruit itself. This is because in general dates have many benefits, some of which can meet the calories and nutrients needed by the body to prevent chronic diseases, improve digestion, maintain immunity, prevent inflammation, and prevent anemia. The lack of studies and analysis related to the addition of date juice to the manufacture of kefir products is an interesting rationale for the author. Based on this description, the author is interested in research to know the effect and know the best concentration of date juice on pH, total lactic acid bacteria, and total yeast in kefir products.

Research is carried out by experimental methods. The experimental design used was a Completely Randomized Design (CRD) with 4 treatments, namely the addition of date juice 10% (P1), 20% (P2), 30% (P3), and 40% (P4). Each treatment was repeated 5 times so that 20 experimental units were obtained. The data obtained from the results of the study were analyzed by Analysis of Variance and if the treatment had a real effect on the variables, it was continued with the Duncan Multiple Region Test (DMRT) at the significance level of α = 0.05. 

1. Materials 

The main ingredient used is kefir grain obtained from the Kefir Bandung store, fresh milk obtained from KPS. Eka Putra Jaya Jatinangor, and sukari dates were obtained from the Alif Date shop Bandung. Other additives used in the analysis were Akuades, Alcohol, Physiological NaCl, deMan Rogosa Sharpe Agar (MRSA), and Malt Extract Agar (MEA). 

1. Methods

Preparation of date juice is done by the modified Al-Farsi (2003) [4] method, starting with the date fruit separated from the seeds and cut into smaller sizes. After that, it is soaked using water with a temperature of 50 - 60°C at a ratio of 1:1 for 30 minutes, then ground to form small granules (juice) and filtered to separate with the pulp. The resulting date juice is pasteurized at 60°C for 30 minutes. The manufacture of kefir products is carried out by the modified Bahar (2008) [5] method, starting with fresh milk measured in volume and preheating at 80°C for 15 minutes, then adding date juice according to treatment, and the temperature is lowered to reach a temperature of 30°C. After that, kefir grain is inoculated with as much as 10% of the milk volume, then stirred and incubated at 30°C for 24 hours. After incubation, it is stirred again and filtered to separate the kefir product from the kefir grain.

1. Variable 

3.1 pH Value

Measurement pH value measurement is carried out based on the procedure AOAC (2005) [6], where the pH meter is calibrated first with pH buffers 4 and 7, then rinsed using akuades and dried using tissues. After that, the electrode is dipped in the sample solution until a stable reading is obtained.

3.2         Total Lactic Acid Bacteria 

The calculation of total lactic acid bacteria was carried out based on the procedure AOAC (1995) [7], with the Total Plate Count (TPC) method on MRSA media. The procedure begins with the sterilization of equipment and media using an autoclave. After that, dilution is carried out using 1 mL of a sample taken and inserted into a test tube containing 9 mL of physiological NaCl then homogenized with a vortex (10^-1). A total of 1 mL of the solution of dilution 10^-1 is taken and put into a test tube containing 9 mL of physiological NaCl (10^-2), the same is done up to a dilution of 10^-7. 1 mL of the solution of dilutions of 10^-5, 10^-6, and 10^-7 is taken and poured into sterile petri dishes. A total of 25 mL of MRSA media is poured into a petri dish containing a solution and homogenized, then let sit until solid and frozen. Then incubated at 37°C for 24 hours with the cup positioned upside down. The total lactic acid bacteria are calculated by the calculation formula:

Description :

N     : Number of colonies (CFU/mL)

ΣC   : Total number of colonies on the counted plate (25 - 250)

n₁    : Number of colonies in the 1^st dilution

n₂                    : Number of colonies in the 2^nd dilution

d              : First dilution counted or meets the conditions (25 - 250)

3.3         Total Yeast 

The calculation of total yeast is carried out based on the procedure AOAC (1995) [7], with the Total Plate Count (TPC) method on MEA media. The procedure begins with the sterilization of equipment and media using an autoclave. After that, dilution is carried out using 1 mL of a sample taken and inserted into a test tube containing 9 mL of physiological NaCl then homogenized with a vortex (10^-1). A total of 1 mL of the solution of dilution 10^-1 is taken and put into a test tube containing 9 mL of physiological NaCl (10^-2), the same is done up to a dilution of 10^-7. 1 mL of the solution of dilutions of 10^-5, 10^-6, and 10^-7 is taken and poured into sterile Petri dishes. A total of 25 mL of MEA media is poured into a petri dish containing a solution and homogenized, then let stand until solid and frozen. Then incubated at a temperature of 25°C for 24 – 48 hours with the cup positioned upside down. Total yeast is calculated by the same formula as the calculation of total lactic acid bacteria.

The results of research on the effect of adding date juice on pH, total lactic acid bacteria, and total yeast kefir products, are presented in Table 1.

Table 1. Summary of Research Results of the Effect of Various Date Juice Treatments on pH, Total Lactic Acid Bacteria, dan Total Yeast in Kefir Products

a–c Different superscripts in the same column represent significant differences (p<0.05).

1. pH Value 

The results of the analysis of variance found that the treatment of adding date juice with different concentrations in the manufacture of kefir products showed a real effect (P<0.05) on the pH value. Based on the data obtained in Table 1. the average pH value of kefir products obtained ranges from 3.70 - 3.80. The pH value of each date juice addition treatment is in accordance with the Food Standards Australia New Zealand (FSANZ) which is set in the pH value of fermented milk beverage products (2.5.3), which is below the value of 4.5 (FSANZ, 2015). 

The results showed that the treatment of adding 10% - 30% date juice concentration was not significantly different, allegedly because the added date juice had a pH of 6.55 so the addition of date juice up to 30% did not cause a significant difference in pH values. In the manufacture of kefir products, there is a decrease in pH from milk pH 6.58 to a lower kefir pH, this condition occurs because, during the fermentation process, there is an increase in the number of lactic acid bacteria accompanied by the formation of lactic acid which causes an increase in acidity so that it can reduce the pH value. This condition is following the opinion of Syukur (2012) [8] that lactic acid bacteria can reduce the pH value of their environment to 4.5 - 3.0. 

Lactic acid bacteria grow well due to the addition of date juice which is used as a prebiotic source for its growth media. The higher the concentration of date juice added, the more lactic acid is formed and causes a low pH value. As Wasilu et al. (2021) [9] argue, the increase in sucrose concentration will be directly proportional to the acidity level because the resulting pH value becomes low. Low pH values can also occur due to the conversion of lactose carried out by lactic acid bacteria into glucose and galactose, these results then continue in the glycolysis pathway to become pyruvic acid. After that pyruvic acid will be converted into a more specific final product in the form of lactic acid, where the results of the overhaul cause a decrease in the pH value of kefir products [10]. 

An increase in pH value in 40% concentration treatment can be influenced by the high concentration of dissolved date juice which can cause changes in osmotic pressure and result in inhibition of the growth of microorganisms in kefir. In accordance with the opinion of Effendi & Parhusip (2021) [11], where the addition of substrates that exceed the limit can cause differences in osmotic pressure in fermentation media, and plasmolysis in microorganism cells so that the breakdown of substrates that become metabolites of organic acids will be inhibited, where these conditions increase the pH value of kefir products. An increase in pH value can also occur because date juice contains saponins and flavonoids that can provide bactericidal effects [12]. Based on these conditions when the total bacteria become lower, the pH value can be higher.

1. Total Lactic Acid Bacteria 

The results of the analysis of variance found that the treatment of adding date juice with different concentrations in the manufacture of kefir products showed a real effect (P<0.05) on total lactic acid bacteria. Based on the data obtained in Table 1. The average total lactic acid bacteria kefir products obtained ranged from 1.78 x 10⁷ CFU/mL - 2.94 x 10⁷ CFU/mL. These results showed that the addition of date juice to kefir had a positive impact on the growth of lactic acid bacteria. This growth occurs because during the fermentation process date juice is added as a source of energy and nutrients for metabolism and the growth of lactic acid bacteria.

The addition of date juice at a concentration level of 30% gives the best results against total lactic acid bacteria, which is 2.94 x 10⁷ CFU/mL because it produces the highest bacterial growth rate in kefir products. The higher concentration of adding date juice will influence the total lactic acid bacteria that are getting higher, where the lactic acid bacteria can utilize date juice as a growth medium. In accordance with the statement of Nofrianti et al. (2013) [13], that there is an increase in the concentration of sugar added, the substrate for microbes will be more and more rapid and its growth will be faster as well so that the activity of bacterial starters in degrading sugar and other organic matter into lactic acid will be higher as well. 

Added date juice can be said to be a source of carbohydrates that help in the process of lactose breakdown by a bacterial starter. This is because carbohydrate compounds in dates contribute to accelerating the growth of lactic acid bacteria, in accordance with the opinion expressed by Lestari et al. (2016) [14], where dates contain sugar which chemically consists of sucrose, glucose, and fructose. Herawati & Wibawa (2011) [15], said that sugar, be it sucrose, glucose, or fructose besides being a source of sweet taste is also a good source of energy for the growth of lactic acid bacteria. 

The decrease in total lactic acid bacteria at 40% concentration treatment can be caused by an increase in dry matter and total solids in kefir so that more date juice substitutions can narrow the space for movement and reduce the growth rate of lactic acid bacteria in breaking down carbohydrates into lactic acid which results in less than optimal growth. This is in accordance with the opinion of Pradipta et al. (2020) [16], who said that the increasing concentration of sucrose will affect changes in the environmental conditions of bacterial growth so that it can reduce total lactic acid bacteria. The increase in total solids occurs because dates have a fiber content of 9.11% [3].

Based on this, in the fermentation process, the dry matter contained in kefir will bind the water content so that the availability of water will be reduced and can inhibit the growth of lactic acid bacteria. This condition can be seen from the texture or viscosity at a concentration of 40% which produces a thicker texture when compared to concentrations of 10% - 30%. The decrease in total lactic acid bacteria is also thought to be due to the treatment of adding date juice to kefir is antimicrobial so it will inhibit the growth of these bacteria. Supported by research by Sani et al. (2017) [12] which shows that dates have antibacterial activity, this happens because of the content of saponins and flavonoids that can form pores on the membrane, thus providing a bactericidal effect. 

The total lactic acid bacteria from each treatment of adding date juice is in accordance with the Codex Standard for Fermented Milk (CODEX STAN 243-2003) which is set in the total lactic acid bacteria standard for kefir, which is at least 10⁷ CFU/mL (CODEX, 2003). Then in accordance with the Standard National Indonesian (SNI 7552: 2018) which is set on drinking fermented milk for lactic acid bacteria, which is a minimum of 10⁶ CFU/mL [17]. The results showed that the activity of bacteria contained in kefir grain can still obtain tolerable environmental conditions for the growth of lactic acid bacteria, so that the total lactic acid bacteria cover the optimal range value. 

1. Total Yeast 

The results of the analysis of variance found that the treatment of adding date juice with different concentrations in the manufacture of kefir products showed a real effect (P<0.05) on total lactic acid bacteria. Based on the data obtained in Table 1. The average total lactic acid bacteria kefir products obtained ranged from 1.78 x 10⁷ CFU/mL - 2.94 x 10⁷ CFU/mL. The results showed that total Yeast was lower when compared to total lactic acid bacteria. This happens because the microbes contained in kefir are more dominated by lactic acid bacteria, in accordance with the opinion expressed by Cho et al. (2018) [18], that kefir fermented using kefir grain contains more than 50 species of lactic acid bacteria, then yeast and acetic acid bacteria.

Yeast contained in kefir will break down simple sugars found in date juice into alcohol and carbon dioxide. This is in accordance with the opinion of Hawusiwa et al. (2015) [19], who said that the glucose content used by yeast is divided into two functions, the first is to grow and multiply, and the second will be converted into metabolite products in the form of alcohol, CO2, and organic acids. Thus, the result of the kefir fermentation process will have a sour taste, then fizzy due to the content of CO₂ and ethanol [20]. In addition, the combination of CO₂ and alcohol fermented by the starter will produce foam on kefir [21].

The lower total yeast can be influenced by the pH and temperature conditions of kefir. Budiyanto (2003) [22] said that the optimum pH value for yeast growth is in the range of 4 - 4.5, while the optimum temperature for yeast growth is 25 - 30°C [23]. A low pH value indicates a high total acid, which causes the yeast to be unable to break down the substrate as it did at the beginning of fermentation. In accordance with the statement of Yusriah & Agustini (2014) [21], where the high total acid content will inhibit the growth process of microorganisms including yeasts that carry out the fermentation process, this condition results in the inability of yeast to break down the substrate as at the beginning of fermentation. 

This condition is supported by Anwar et al. (2012) [24], where pH that is too acidic or alkaline and CO₂ concentrations that are too high will be inhibitor agents in yeast growth. These inhibitor agents can cause yeast growth to be less optimal because yeast is included in the type of aerobic microbes where oxygen needs to support life. The results showed suitability when compared to these conditions, where the low pH value in the treatment of 10% - 30% indicates a low total yeast as well, while an increase in the pH value will indicate an increased total yeast. As happened in the 40% treatment, there was an increase in total yeast from 1.16 x 10⁷ CFU/mL - 1.76 x 10⁷ CFU/mL. 

The total yeast from each treatment of adding date juice is in accordance with the Codex Standard for Fermented Milk (CODEX STAN 243-2003) [25] which is set at the total yeast or yeast standard for kefir, which is at least 10⁴ CFU/mL. These results show that yeast activity contained in kefir grain can still obtain tolerable environmental conditions for yeast growth, resulting in a total yeast average that is in accordance with standards.

The addition of date juice with different concentrations to kefir products affects the pH value, total lactic acid bacteria, and total yeast. The addition of date juice with a concentration of 30% in the manufacture of kefir products gave the best results with a pH value of 3.72, total lactic acid bacteria 2.94 x 10⁷ CFU/mL, and total yeast 1.16 x 10⁷ CFU/mL. 

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

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