1. Introduction
Probiotics have been known for centuries, due to their amazing health benefits. In this context, researchers have regard to probiotic studies relating to fermented milk products. Moreover, consumers prefer to ingest such foods as an alternative to conventional therapies for various chronic diseases. Kefir is one such probiotic product; it is a traditional, acidic, fermented probiotic dairy product that originated in the Caucasus Mountains thousands of years ago. It is made by fermenting any type of pasteurized milk with starter culture or kefir grains [
1,
2,
3]. In addition to beneficial bacteria and yeast, kefir contains vitamins, minerals and essential amino acids which aid the human body in hemostasis, regulation and healing mechanisms [
4,
5]. Kefir has a wide spectrum of health benefits including physiological, prophylactic and therapeutic properties. These effects are due to the presence of a wide variety of bioactive compounds produced during the fermentation process, and the highly diverse microbial content, which act either independently or synergistically to influence these health benefits [
6].
Consumer demand is continuously increasing for natural ingredients that possess specific functional properties and that improve the nutritional value of dairy products. For example, the addition of flaxseed mucilage (FM) and gum arabic (GA). FM is a plant extract obtained from flaxseed; it has a positive effect on the growth of probiotic bacteria. Flaxseed is a member of the
Linaceae family known as Linseed (
Linum usitatissimum L.) and it has two basic varieties: brown and yellow [
7]. It is considered to be rich in nutrients such as flavonoids, minerals, vitamins, and carbohydrates, which contribute to many potential health benefits. It also contains 20% protein, 7.7% moisture, 3.4% ash, 30–40% oil, as well as alpha-linolenic acid (ALA), which is a precursor of omega-3 fatty acids and which has a positive effect on the growth of
B. lactis [
8]. Moreover, flaxseed contains 35–45% fiber, of which about two-third is insoluble and one third is soluble. This fiber is mucilage [
9].
Mucilage is a jelly-like substance that is responsible for holding water in plants, making them drought-resistant. It is extracted by placing seeds into water, and is then filtrated [
10]. Flaxseed mucilage can be used as a food gum due to its rheological properties such as thickening, emulsification, and gelling. Mucilage is used as a food additive by food manufactures [
11]. Mucilage has many practical uses in the modern world such as: treating burns, wounds, ulcers, irritation, diarrhea, constipation, diabetes and cardiovascular disease; protection against colon cancer; treatment of obesity; and many other beneficial effects [
12].
Gum arabic (GA, E-Number 414) is an edible, dried, gummy exudate from the stems and branches of
Acacia senegal and
A. seyal that is rich in non-viscous soluble fiber [
12]. Many researchers have studied the effect of gum arabic and other types of gums on the growth and activity of probiotic bacteria [
13,
14,
15,
16].
Consumers’ interest in healthy diets and wellness has led to an increase in the consumption of foods containing probiotics and prebiotics. Prebiotics are non-digestible carbohydrates that reach the colon, where they are selectively fermented, stimulating the growth and/or activity of one or a limited number of beneficial bacteria. Probiotics and prebiotics, or combinations thereof, are choice in new food developments due to their ability to improve gut health and body comfort. According to previous research, one probable method to enhance the growth and firmness of probiotic bacteria is to fortify dairy products with prebiotics [
17].
Researchers used gums and polysaccharides as a prebiotic source, to investigate their effect on the survival of probiotics, and found that they could improve the survival of probiotics. When prebiotics and probiotics are used together, they show symbiotic behavior and provide beneficial effects to the host [
18,
19]. Also, some researchers observed that flaxseed could increase the viability of probiotics and improve some other properties of the product [
20,
21]. To the best of our knowledge there have been no studies about the new properties and efficacy of kefir after adding GA and FM in different concentrations in order to determine the symbiotic effect of this combination on
Lactobacillus acidophilus and
Bifidobacterium lactis counts within 28 days of storage. Nor have there been any studies to show how FM and GA can affect pH, viscosity, titratable acidity, color, protein and the total solid values of kefir during storage time.
2. Materials and Methods
2.1. Extraction of Flaxseed Mucilage
About 100 g flaxseed was made up to 2 L with distilled water in a glass beaker and stirred by magnetic stirrer (Ms300Hs, Mtops, Seoul, Republic of Korea) for 3 h (at 55 °C and 60 rpm). Then, the FM was filtrated from the seeds using muslin cloth. The resulting liquid, mucilage, was distributed in small quantities (about 150 mL) into small pyrex glass dishes and put in a forced convection oven (JSOF-100, JS Research Inc. Gongju-City, Republic of Korea) overnight at 55 °C. Then, the dried mucilage (moisture content 3.2%) was collected and stored at 4 °C up to usage.
2.2. Experimental Design
Raw cow’s milk was pasteurized at 90 °C for 5 min and cooled down to 25 °C. pH of milk was recorded as 6.22. Then kefir (
Lactobacillus kefiranofaciens,
Lactobacillus acidophilus,
Lactobacillus casei,
Lactobacillus reuteri,
Lactobacillus plantarum,
Streptococcus thermophiles,
Leuconostoc mesenteroides and at least 1 × 10
8 log CFU/ mL viable probiotic bacteria) and yogurt starter culture (
Lactobacillus bulgaricus and
Streptococcus thermophiles) were inoculated as 0.02% (
w/
v), 0.03% (
w/
v), respectively. After that,
Bifidobacterium lactis was added into the mixture as 0.06% (
w/
v) after activation at 30 °C for 7 h. The mixture was divided into 9 parts (
Table 1). Thereafter, FM was added at a concentration of 0.03, 0.05, and 0.1% (
w/
v) where GA was added into each mixture at 0.2% (
w/
v). Each part was stirred with magnetic stirrer (Ms300Hs, Mtops, China) for 20 min to dissolve FM and/or GA. Next, each 9 group samples divided into 6 according to each storage time, defined as 0, 1st, 7th, 14th, 21th and 28th days. Then, all samples were put in an incubator (Nüve ES 500, Turkey) at 25 °C for 24 h up to the pH value reached to 4.0. After that, each of the samples was stored at refrigerator at 4 °C for 28 days. All parameters were measured at the sampling time of 0, 1st, 7th, 14th, 21st and 28th days in duplicate.
2.3. pH and Acidity
The pH values were recorded using a pH meter (pH/mV/Cond./TDS/Temp. meter 86505), on the 0, 1st, 7th, 14th, 21st and 28th days, at a temperature of 25 °C.
Acidity was measured by adding 3 drops of phenolphthalein to each sample and titrating it with 0.1 NaOH. The % acidity was measured and calculated as lactic acid% [
22].
where v is the volume of titrant and m is the weight of the sample.
2.4. Viscosity
The viscosity of kefir was measured by a viscometer (Brookfield, DV3T™ viscometer, Worcester County, MA, USA). Viscosity measurement was carried out using V-72(72) spindle of 30 rpm at constant temperature (25 °C). Viscosity was recorded after 25 s and 250 mL sample was used for each run [
22]. Viscosity data were expressed in cp.
2.5. Color
The colors (L*, a* and b*) of all kefir samples were measured using Hunter lab ColorFlex (A60-1010-615 Model colorimeter, Hunter lab, Reston, VA, USA) on the 0, 1st, 7th, 14th, 21st, and 28th, days of storage [
22]. Each time, white and black ceramic plates were used for standardization of the instrument (L
0 = 93.01, a
0 = −1.11, and b
0 = 1.30). The Hunter L, a, and b values correspond to lightness, greenness (−a) or redness (+a), and blueness (−b) or yellowness (+b), respectively. The color measurements were performed at room temperature (25 ± 2 °C) in duplicate.
2.6. Microbial Analysis
The enumerations of specific lactic acid bacteria were specified as log CFU/mL. Lactic acid bacteria counts were conducted using de Man, Rogosa and Sharpe (MRS) enriched agar. MRS agar was enriched with 1% maltose and 1% raffinose for
Lactobacillus acidophilus and
Bifidobacterium lactis counts, respectively. All kefir samples were diluted tenfold using peptone water (0.2%,
v/
v), then spread on MRS enriched agar and incubated at 30 °C and 5% CO
2 for 72 h (3 days). Then, the numbers of colonies were calculated. Enumerations of bacteria were conducted in duplicate on petri dishes. The counts of bacteria in kefir fortified with 0.2% (
w/
v) GA with 0.03, 0.05, 0.1% (
w/
v) of FM, 0.03% (
w/
v) yoghurt starter culture and 0.06% (
w/
v)
Bifidobacterium lactis were recorded on the 0, 1st, 7th, 14th, 21st and 28th days of storage. The counts of
Lactobacillus acidophilus and
Bifidobacterium lactis were recorded according to Dave and Shah [
23].
2.7. Total Solid and Protein Analysis
Total solid content was quantified using the oven method prescribed by the Turkish Standards Institute (TSI) [
22]. Approximately 3 g of kefir (ws) was placed in a pre-weighed (w
1), pre dried small glass dish and transferred to a hot air oven at 105 °C up to constant weight reached. Samples were cooled in a desiccator before final weights were recorded (w
2).
Protein contents of samples were determined according to the TSI [
24]. Ten milliliters of sample, 0.5 mL of 0.5% phenolphthalein indicator and 0.4 mL of neutral saturated potassium oxalate were mixed in a conical flask. Then, the mixture was neutralized with 0.1 M NaOH until getting a standard pink color. After that, 2 mL of formalin (37% formaldehyde) was added and titrated again (a). Two milliliters of formalin and 10 mL of water were titrated separately with the same alkali (b) as blank. The protein content of the sample was calculated as follows [
24]:
2.8. Sensory Analysis
Hedonic sensory analysis was performed by fifteen trained assessors (graduate students in Gaziantep University Food Engineering Department) to estimate the kefir samples according to the following characteristics: overall appearance (rheology), color, taste/flavor, smell/odor, thickness. Scores were given to each sample as follows: liked-3, normal-2, dislike-1 according to the standard TS EN ISO 8589 [
25].
2.9. Statistical Analysis
The differences between samples were assessing using one-way ANOVA to compare probiotic bacteria counts, pH, acidity, viscosity and colors under different conditions. Statistical analyses were carried out using the SPSS statistical package version 26.0 (IBM Corporation, Armonk, NY, USA). The Duncan multiple range test was performed to find a statistically significant group at α = 0.05 level. Pearson’s correlation was also applied for correlation analysis. It was used to determine the correlation coefficients between the microbial counts (L. acidophilus and B. lactis), pH, acidity, viscosity and color parameters (L*, a*, b*).
4. Conclusions
In this study, the addition of 0.03, 0.05 and 0.1% FM alone, or mixed with 0.2% GA, into kefir affected the counts of viable L. acidophilus, B. lactis, pH value, acidity, viscosity, color and protein value on the 0, 1st, 7th, 14th, 21st and 28th days of storage. The FM significantly improved the survival of L. acidophilus and B. lactis, as well as affecting the other physicochemical properties of the kefir. Therefore, it could be concluded that FM has good potential to improve kefir’s quality in respect of its health benefits. Although it could result in lower sensory scores compared to the control, it is a good option as a complementary food for human health in general, and in particular for people who suffer from gastrointestinal problems and autoimmune weakness.