Functional properties of cow's milk and soy drinks prepared by fermentation with probiotic and yoghurt bacteria

Abstract

Fermented probiotic products deserve special attention because they have probiotic activity and unique nutritional, sensory, and therapeutic properties, which is why they are classified as functional products. Since both probiotics and soy beverages are beneficial to human health, this study aimed to evaluate the chemical and functional characteristics of fermented beverages based on cow’s milk and soy beverage in addition to probiotic cultures. The content of macro and microelements in the produced beverages, polyphenolic components as well as the content of isoflavones were monitored. Having in mind the importance of minerals in the diet, it can be said that all variants of fermented beverages are a very rich source of calcium, potassium, and sodium. Based on the results of physical and chemical changes in the quality of fermented dairy beverages made from a mixture of cow's and soy beverage, concerning the characteristics of the products obtained by using only cow's milk, can be explained by the justification of the use of soy beverage in the production of fermented dairy products, with the aim of obtaining new functional fermented dairy product with a high content of bioactive components, mineral elements, significant nutritional and organoleptic properties.

Keywords:
L.acidophilus; L.casei; minerals; phenolic compounds; isoflavones

1 Introduction

Functional food is the most promising field of nutritional science. This food is interesting from the consumer point of view with the prospect of maintaining health and preventing diseases using natural food as part of a regular diet. Several raw materials can be used for healthy purposes and soybeans are among those with the greatest potential (Šertović at al., 2012).

Numerous studies conducted in the last 20-30 years have shown that soy, and thus to some extent (depending on the method of preparation) and soy protein products, are a good source of biologically active compounds (often called phytochemicals) such as many biologically active protein, phytosterols, isoflavones, saponins, and others. Soy milk is a good substrate for the growth of lactic acid bacteria, and especially bifidobacteria. Fermentation of soy milk provides the ability to transform and improve flavor and texture (Liu, 1997; Tsangalis et al., 2003). Also, the fermentation of soy milk is considered a good substrate for the development and production of functional foods. It is the fermentation of soy milk by probiotic bacteria that reduces the level of oligosaccharides and raises the level of free isoflavones (Wei et al., 2007). Lactobacillus acidophilus and Lactobacillus casei are considered to be the most important probiotic organisms and are believed to exert positive effects on human health (Parra et al., 2013).

Fermentation of soy milk with probiotic bacteria also improves the nutritional value of these products and allows food to function as delivery of probiotic organisms to consumers (Tratnik & Božanić, 2012). Although the concentration of selected major and toxic elements has been studied in some soy-based formulations, little research has been done on the mineral content of soy yogurt and yogurt produced from different ratios of cow’s milk and soy beverage. So, this study was conducted to investigate the assessment as well as to determine the effect of the combination of cow's milk and soy beverage and probiotic bacteria on the content of polyphenolic compounds, isoflavones, and mineral components in fermented probiotic drinks based on soy.

2 Material and methods

Homogenized, UHT sterilized cow’s milk with 2.50% fat (Meggle, Bihać, B&H) and soy beverage with 1.90% fat, brand dmBio (GmbH + Co.KG, Germany) were used to produce probiotic drinks. The physical, chemical and microbiological characteristics of milk samples were following the standards. Yogurt culture YF-L811 (Christian Hansen, Horsholm, Denmark) and the probiotic strain La5 and Lc01 (Christian Hansen, Horsholm, Denmark) were used for the fermentation of different mixtures of cow's milk and soy beverage.

2.1 Production of fermented beverages

Five different proportions of homogenized permanent milk into soy beverage (100:0, 75:25, 50:50, 25:75, 0:100) were prepared by mixing permanent UHT milk and soy beverage. Samples were inoculated with probiotic starter cultures (La5 and Lc01) in addition to yogurt culture. Probiotic monocultures for inoculation were in lyophilized DVS (Direct Wat Set) form. An inoculum was first prepared by inoculating 0.1 g of probiotic cultures and 0.07 g of yogurt culture into 100 mL of milk. A separate inoculum was made for each of the five different sample ratios and incubated for 30 minutes at 43 °C to allow the bacteria to adapt to the medium. After incubation, the inoculum was inoculated into milk samples intended for the production of probiotic beverages (Šertović et al., 2019). The fermentation was carried out at 43 °C until the pH reached 4.6. The characteristics of the produced probiotic milk beverages were monitored at the end of fermentation. After production, the samples were stored in a refrigerator at + 4°C. Three repetitions of fermentation of a mixture of cow's milk and soy beverage were performed.

2.2 Determination of mineral elements by ICP-OES method

0.6 to 0.7 g of the lyophilized sample of produced fermented beverages was treated with a mixture of 7 mL of 65% HNO3 and 1 mL of 35% H2O2 (Zorka, Sabac) and transferred to special PTFE vials for digestion. Microwave digestion (ETHOS 1; Mileston, Bergamo, Italy) was used for the sample mineralization process. The final clear solution was transferred to normal 50 mL vessels and made up to the line with ultrapure water. The blank was prepared in the same way but without the yogurt sample. Minerals and trace elements were determined using the induced coupled plasma optical emission spectroscopy (ICP-OES; ICAP 6500 Duo ICP, Thermo Scientific, UK) method. The results are expressed as mg of mineral in 100 g of yogurt sample.

2.3 Analysis of polyphenolic components content in fermented milk beverages

Preparation of standard solutions

A stock solution of a mixture of all polyphenolic standards and cis, trans-abscisic acid was made by dissolving in methanol (HPLC purity) so that each had a concentration of about 1000 mg/L in the mixture. By diluting the stock solution with the mobile phase, a series of working solutions of the following concentrations was obtained: 0.025; 0.050; 0.100; 0.250; 0.500; 0.750; 1,000 mg/L. The stock and working solutions were stored in the dark at 4 °C until analysis. Calibration curves were obtained by correlating the surface of the peaks with the concentration of standard solutions.

Quantitative analysis of polyphenolic compounds

Separation and quantification of polyphenols in fermented beverage samples was performed on a Dionex Ultimate 3000 HPLC system equipped with a DAD detector connected to a TSQ Quantum Access Max mass spectrometer with a detector with three analyzers - triple quadrupole. Elution was performed at 40 °C on a Syncronis C18 assay column. The mobile phase consisted of water + 0.1% formic acid (A) and acetonitrile (B), which were in the following gradient concentrations: 5% B, 2.0 min; 5–95% B, 1.0–16.0 min; 95–5% B, 16.0–16.2 min and 5% B up to 20 min. The flow was adjusted to 0.3 mL/min and the wavelengths to 254 and 280 nm. The injection volume was 5 µL. The TSQ Quantum Access Max mass spectrometer was equipped with an ion source in the form of electrospray ionization at a temperature of 200 °C. The spray voltage was 5 kV and the capillary temperature was 300 °C (Gašić et al., 2015). The mass spectrometer recorded masses in a negative mode in the range of 100 to 1000 m/z. To quantify the polyphenols for each standard, a molecular ion and the two most intense fragments from the MS2 spectrum were recorded separately. Polyphenols have been identified by comparison with commercial standards. The total content of each compound was calculated by integrating the peak areas and expressed as mg/kg.

2.4 Statistical analysis of the results

The results of the analyzed samples are presented as the mean value of repetition ± standard deviation. One-way analysis of variance (ANOVA) and multiple comparisons (Duncan's post-hoc test) were used to assess a significant difference in data at a significance level of p < 0.05. Statistics were performed using Microsoft Office 2014 and a demo version of the statistical package for MS Office XLSTAT-Pro 2014. Principal Components Analysis (PCA) was also performed.

3 Results and discussion

3.1 Macro and micro elements in dairy products by ICP-OES method

To assess the nutritional value of different samples of fermented cow's milk and soy beverages, the content of 18 macro-and micro-elements was analyzed, and the results are shown in Tables 1 and 2. Based on the results shown in Table 1, it can be concluded that the potassium content in all ratios of fermented beverages was the highest of all tested macronutrients (K, Ca, Mg, Na, P, and S). The higher amount of potassium in the samples of fermented beverages in relation to other macroelements is not unknown, because potassium is one of the most important macroelements, which is very important for membrane transport, energy metabolism, and normal cell functioning.

Table 1
Concentration (mg/L) of macroelements in samples of probiotic beverages at the end of fermentation.

Mineral	Calcium	Magnesium	Phosphorus	Sodium	Potassium	Sulfur
Samples	mg/L
1A	892.8 ± 44.7a	78.66 ± 3.1g	720.84 ± 8.2c	280.02 ± 3.6a	1112.07 ± 61.2h	258.39 ± 3.5i
2A	700.28 ± 47.5bc	105.37 ± 7.6e	739.92 ± 25,2a	218.89 ± 9.1c	1141.26 ± 132.6f	264.42 ± 9.3g
3A	564.01 ± 26.08c	134.38 ± 11.36c	736.20 ± 3.18b	188.24 ± 8.44c	1313.46 ± 43.10c	290.94 ± 2.61c
4A	375.89 ± 20.9d	161.11 ± 18.22b	628.42 ± 18.5d	169.54 ± 1.69d	1455.60 ± 5.27b	312.36 ± 9.37b
5A	149.58 ± 6.87e	195.57 ± 10.19a	528.63 ± 11.8e	141.80 ± 3.56e	1808.45 ± 47.50a	340.71 ± 8.23a
1C	775.84 ± 16.9b	74.69 ± 0.9h	692.35 ± 25.1c	225.16 ± 6.1b	978.93 ± 8.8i	245.34 ± 9.50j
2C	689.23 ± 56.3c	98.54 ± 8,69f	678.93 ± 10.3cd	215.47 ± 17.5d	1059.23 ± 85.2g	256.02 ± 2.2h
3C	561.28 ± 40.07d	125.52 ± 10.2d	649.43 ± 9.6d	202.56 ± 0.4e	1324.24 ± 77.2e	300.56 ± 2.6e
4C	390.42 ± 45.5e	159.35 ± 9.8b	595.54 ± 18.8e	187.77 ± 7.5f	1581.83 ± 76.6c	317.20 ± 7.3c
5C	138.38 ± 6.3g	181.31 ± 5.8ab	535.45 ± 15.7f	137.88 ± 3.5i	1654.91 ± 96.5b	343.52 ± 15.1a
Mean YA	536.51 ± 287.2A	135.02 ± 10.09A	670.80 ± 6.4A	199.69 ± 3.3A	1366.16 ± 46.6A	293.36 ± 3.3A
Mean YC	511.03 ± 253.8A	127.88 ± 7.07A	630.34 ± 6.4A	193.76 ± 6.5A	1319.82 ± 34.5A	292.52 ± 5.3A
Total Mean	523.7 ± 270.5	131.45 ± 8.58	650.57 ± 6.4	196.72 ± 4.9	1342.99 ± 40.5	292.94 ± 4.3

• 1A-100% cow's milk; 2A-75% cow's milk + 25% soy beverage; 3A-50% cow's milk + 50% soy beverage; 4A-25% cow's milk + 75% soy beverage; 5A-100% soy beverage; A – L. acidophilus; 1C-100%cow'milk; 2C-75% cow's milk + 25% soy beverage; 3C-50% cow's milk + 50% soy beverage; 4C-25% cow's milk + 75% soy beverage; 5C-100% soy beverage; C - L.casei. Data represent mean values (± SD) of three replicates. ^abcDuncan's test confirmed a statistically significant difference between mean values (± SD).

Table 2
Concentration (mg/L) of trace elements in samples of fermented products at the end of fermentation.

Mineral	Barium		Copper	Chromium	Zinc		Iron		Manganese
Samples					mg/L
1A	0.05 ± 0.007c		0.06 ± 0.008e	0.03 ± 0.008a	2.66 ± 0.23e		0.52 ± 0.054e		0.028 ± 0.004e
2A	0.04 ± 0.002e		0.29 ± 0.045d	0.007 ± 0.0075cd	2.90 ± 0.093d		1.54 ± 0.09d		0.335 ± 0.029d
3A	0.04 ± 0.002d		0.55 ± 0.022c	0.006 ± 0.0018d	3.10 ± 0.047c		2.88 ± 0.27c		0.667 ± 0.068c
4A	0.06 ± 0.004b		0.77 ± 0.007b	0.008 ± 0.0093cd	3.38 ± 0.0035b		4.25 ± 0.067b		1.01 ± 0.044b
5A	0.09 ± 0.006a		1.13 ± 0.008a	0.017 ± 0.003b	3.65 ± 0.051a		6.35 ± 0.35a		1.54 ± 0.083a
1C	0.03 ± 0.02e		0.05 ± 0.02i	0.01 ± 0.02c	2.51 ± 0.12h		0.76 ± 1,5i		0.77 ± 0.008g
2C	0.06 ± 0.003c		0.21 ± 0.01g	0.008 ± 0.004e	2.69 ± 0.03f		1.49 ± 0.2h		0.37 ± 0.003e
3C	0.06 ± 0.005c		0.48 ± 0.02e	0.009 ± 0.004d	3.05 ± 0.04d		3.10 ± 0.12e		0.66 ± 0.04d
4C	0.07 ± 0.005b		0.77 ± 0.03c	0.01 ± 0.02c	3.38 ± 0.12c		4.94 ± 0.5c		1.09 ± 0.05c
5C	0.09 ± 0.006a		1.11 ± 0.04b	0.01 ± 0.01c	3.47 ± 0.07b		7.58 ± 1.9a		1.57 ± 0.03a
Mean YA		0.056 ± 0.002A	0.56 ± 0.02A	0.014 ± 0.003A		3.14 ± 0.09A		3.11 ± 0.13A	0.716 ± 0.03A
Mean YC	0.062 ± 0.02A		0.52 ± 0.43A	0.01 ± 0.001A	3.02 ± 0.001A		3.57 ± 0.81A		0.892 ± 0.45A
Total Mean	0.059 ± 0.013A		0.54 ± 0.29A	0.01 ± 0.002A	3.08 ± 0.06A		3.34 ± 0.48A		0.81 ± 0.29A
Mineral	Aluminium		Lead	Cobalt	Cadmium		Nickel		Selen
Samples
1A	N.D.		0.006 ± 0.02b	0.002 ± 0.004f	0.01 ± 0.002g		0.04 ± 0.007e		0.04 ± 0.07c
2A	0.07 ± 0.026d		0.002 ± 0.007e	0.0001 ± 0.004h	0.006 ± 0.001b		0.09 ± 0.017d		0.05 ± 0.02b
3A	0.09 ± 0.015c		0.002 ± 0.008e	0.003 ± 0.004e	0.004 ± 0.001d		0.21 ± 0.003c		0.04 ± 0.05c
4A	0.22 ± 0.10b		0.01 ± 0.005b	0.006 ± 0.003b	0.005 ± 0.002c		0.26 ± 0.019b		0.03 ± 0.02d
5A	0.24 ± 0.026a		0.004 ± 0.007c	0.009 ± 0.003a	0.007 ± 0.002a		0.32 ± 0.009a		0.05 ± 0.06b
1C	N.D.		0.008 ± 0.006a	0.001 ± 0.003g	0.002 ± 0.0003f		0.02 ± 0.007i		0.02 ± 0.04e
2C	0.11 ± 0.09e		0.004 ± 0.01c	0.003 ± 0.004e	0.003 ± 0.002e		0.05 ± 0.01g		0.05 ± 0.06b
3C	0.03 ± 0.02h		0.002 ± 0.008e	0.004 ± 0.009d	0.005 ± 0.001c		0.13 ± 0.008e		0.06 ± 0.07a
4C	0.17 ± 0.11d		0.002 ± 0.01e	0.005 ± 0.008c	0.007 ± 0.002a		0.21 ± 0.01d		0.05 ± 0.10b
5C	0.29 ± 0.10a		0.005 ± 0.007bc	0.009 ± 0.004a	0.007 ± 0.0002a		0.30 ± 0.01b		0.03 ± 0.07d
Mean YA	0.15 ± 0.04A		0.005 ± 0.006A	0.004 ± 0.0005A	0.006 ± 0.0005A		0.184 ± 0.007A		0.042 ± 0.023A
Mean YC	0.15 ± 0.04A		0.0042 ± 0.002A	0.004 ± 0.003A	0.005 ± 0.0008A		0.142 ± 0.001A		0.042 ± 0.021A
Total Mean	0.15 ± 0.04		0.005 ± 0.003	0.004 ± 0.002	0.006 ± 0.0002		0.163 ± 0.004		0.042 ± 0.001

• 1A-100% cow's milk; 2A-75% cow's milk + 25% soy beverage; 3A-50% cow's milk + 50% soy beverage; 4A-25% cow's milk + 75% soy beverage; 5A-100% soy beverage; A – L. acidophilus;
• 1C-100%cow'milk; 2C-75% cow's milk + 25% soy beverage; 3C-50% cow's milk + 50% soy beverage; 4C-25% cow's milk + 75% soy beverage; 5C-100% soy beverage; C - L.casei. Data represent mean values (± SD) of three replicates. ^abcDuncan's test confirmed a statistically significant difference between mean values (± SD).

Of the other macronutrients (Ca, Mg, Na, P, and S), calcium was most present, followed by sulfur, phosphorus and sodium, while magnesium was least present in all tested samples of fermented beverages (Table 1). A similar trend has been observed in previous studies (Sa’nchez-Segarra et al., 2000; Sola-Larrañaga & Navarro-Blasco, 2009; Khan et al., 2014; Etiosa et al., 2017). Calcium gives bone strength, while its ions play an important role in many metabolic processes. Lack of calcium in the body can lead to some diseases such as osteoporosis. The recommended daily dose of calcium is 200-1300 mg, depending on human age (Petrović et al., 2016). The acidification process during sample production significantly affects the distribution of calcium. Calcium is a mineral that is present in higher concentrations in cow's milk compared to soy beverages. Therefore, in the analyzed samples, calcium concentrations were higher in the samples that contained a higher amount of cow’s milk compared to the soy beverage. Calcium bound to casein micelles is released during the fermentation process, which leads to an increase in the concentration of the free form. Also, calcium is known as an anticancer agent (Shin et al., 2002), while magnesium is required for insulin activity and is essential for the prevention of type 2 diabetes (Huerta et al., 2005). When it comes to potentially toxic trace elements (Al, Cd, Pb, Sb, Sn) their concentration was low, and some were not even detected in fermented beverages, except Al, which was measured from 0.007 mg/100 g to 0.24 mg/100 g. However, it has been previously described that dairy and cereal products make up about 60% of total Al intake and these levels do not pose any danger to human health (World Health Organization, 1996). Among the content of nutrients (Cu, Fe, Ni, Mn, and Zn), zinc and iron showed the highest level in all samples of fermented beverages. Iron is an essential component of hemoglobin and participates in several oxidation-reduction reactions (Achanta et al., 2007), while zinc is very important because it is one of the trace elements, required for normal growth. It has been proven that there was a dramatic improvement in acrodermatitis enteropathica after the addition of zinc (Petrović et al., 2016), which is of great importance for human metabolism. Zinc can act antiarthritic, anti-infective, antiviral, astringent, immunostimulatory, and as a healing agent, and in some cases, the appearance of acne on the skin can be a symptom of zinc deficiency in the body. Zn and Cu concentrations must be monitored and compared with the values set by the EU and WHO. According to the literature data for cow's milk, the average values of Cu and Zn are 0.43 mg/L and 4.96 mg/L, respectively (Petrović et al., 2016). Based on the results of the content of essential elements in the tested samples of fermented beverages, it can be concluded that different ratios of cow's milk and soy beverage can be a good additional source of iron. In the tested samples, the highest proportion of iron was recorded in the sample of 100% soy beverage (5A-7.58 mg/L, 5C-6.58 mg/L). Iron, which is present in the body, is used for the activity of some energy-producing enzymes, and its deficiency leads to microcytic and hypochromic anemia (Petrović et al., 2016). The presence of copper was also detected in all tested samples of fermented beverages. The copper content was higher in samples that contained higher amounts of soy beverage compared to cow’s milk. Copper is an essential metal in the human body, animals, and plants, and it appears in the form of an element or mineral. It is necessary for the production of hemoglobin and is an important ingredient in a large number of enzymes. As can be seen from Table 2, the main differences between probiotic products of cow’s milk and soy beverage were observed with Ni and fewer nutrients (Cu, Fe, Mn, and Zn). Differences in nutrient levels are visible in Cu and Mn, which appeared at much higher levels in samples from pure soy beverage or in combinations of cow's milk and soy beverage. The total intake of nickel in the human diet varies depending on the amount and proportion of food of animal origin (low nickel content) or plant (high nickel content) origin, as well as the amount of refined or processed food in the diet. Some reports indicate an intake of about 150 μg per day. Taking into account the recommendations of the World Health Organization (WHO), the level of toxicity threshold is set at less than 600 μg/day (Llorent-Martínez et al., 2012). The average level of nickel found in the samples of manufactured beverages ranged from 0.02 to 0.32 mg/L. Similar results were also found in milk samples. Considering the weight of one yogurt (approximately 125 g), Ni intake would be 23.37 μg and 397.5 μg when consuming one milk or soy yogurt. As a result, although no health risk arises from these data, it is clear that routine analysis of trace elements is recommended for soy-produced foods.

Many other trace elements such as lead, cadmium, cobalt, aluminum and selenium also occur in milk and dairy products but are not nutritionally important. Of all the trace elements present in milk samples, aluminum was detected at the highest level. The total aluminum content in fermented samples is affected by many different factors. First of all, aluminum is naturally present in water, and can also be present in food. In the case of a pasteurized cow’s milk sample, there is a possibility that aluminum is delivered through packaging material made of aluminum (Abdolmohammad-Zadeh & Sadeghi, 2010). As regards toxic metals, in particular lead, Commission Regulation (EC) No 1881/2006 set the maximum level for Pb in yogurt at 0.02 mg/kg (EC, 2006). In this study, the mean Pb content in yogurt samples varied between 0.008 mg/L and 0.002 mg/L. Also, the presence of lead was detected in milk samples. This can be the result of various factors such as the water used in the preparation of the product, in the production process, and in the diet. FAO/WHO has defined acceptable daily intake of so-called heavy metals such as 25 mg/kg of weight (Petrović et al., 2016). Therefore, bearing in mind that once absorbed into the bone, liver, or kidneys, this metal has detrimental effects (Hague et al., 2008), it is necessary to monitor and control its content. European legislation does not set a limit for Cd in yogurt. Therefore, the metal content may vary depending on the physical and chemical techniques used in the production of yogurt. The content of mean levels of essential and toxic metals in the yogurt samples obtained in this study was compared with the results of other authors. The metal content of fermented milk according to research varies in many countries (Musaiger et al., 1998; Park, 2000; Güler, 2007; Ayar et al., 2009; Crivineanu et al., 2009; Sanal & Güler, 2010; Llorent-Martínez et al., 2012; Zamberlin et al., 2012). It can be concluded that the concentrations of minerals in fermented beverages are conditioned by the composition of the raw material as well as the technological process used in dairy processing of products. Consumption of yogurt does not lead to a significant intake of toxic metals; therefore, there is no toxicological risk from the daily consumption of these dairy products. This research is a contribution to the current knowledge about the content of essential and toxic metals in yogurt, providing data on their safety and quality. The mineral content results of the analyzed samples of fermented beverages are in accordance with the literature data of De la Fuente et al. (2003). Having in mind the importance of minerals in the diet, it can be said that all variants of fermented dairy beverages are a very rich source of calcium, potassium, and sodium. According to the obtained results, the produced dairy beverages of cow's milk and soy beverage have a significantly more favorable content of tested minerals after production, which gives the advantage of using soy beverage in relation to cow's milk.

3.2 Analysis of polyphenolic compounds content in fermented beverages

Due to their chemical structure, flavonoids are phenol compounds with the highest antioxidant activities. Among dietary flavonoids, isoflavones, especially daidzein and genistein, show one of the highest antioxidant activities (Heim et al., 2002). The content of total and individual polyphenolic compounds and isoflavones detected in samples of fermented cow's milk and soy beverage are shown in Tables 3 and 4. Phenolic acid varies depending on the ratio of soy beverage in cow's milk. The analysis of the content of individual polyphenolic components showed that by increasing the amount of soy beverage in cow's milk, the amount of present phenolic compounds also increased. Quercetin is the most abundant flavonoid present in soy. In all samples prepared from a “mixture” of cow's milk and soy beverage, as well as in samples from pure soy beverage, two main isoflavonoid compounds were detected, namely daidzein and genistein.

Table 3
Content of polyphenolic compounds (mg/kg) in samples of fermented probiotic beverage of cow's milk and soy beverage.

Content of polyphenolic components (mg/kg)	L.acidophilus				L.casei
	2A	3A	4A	5A	2C	3C	4C	5C
GC	n.d.	n.d.	n.d.	0.15a	n.d.	n.d.	n.d.	0.04b
C	n.d.	n.d.	n.d.	0.02b	n.d.	n.d.	n.d.	0.06a
CoA	n.d.	0.40c	1.50b	3,10a	n.d.	n.d.	n.d.	n.d.
QUE	0.007e	0.01d	0.03b	0.06ab	0.005d	0.02c	0.03b	0.07a
EA	0.30d	0.40c	0.50b	0.9a	n.d.	0.20f	n.d.	0.20e
API	0.007d	0.02c	0.03b	0.04a	0.009d	0.02c	0.02c	n.d.
SNA	n.d.	0.50c	n.d.	n.d.	n.d.	2.30b	n.d.	6.80a
Myn	0.004c	0.007b	n.d.	0.01a	0.002c	0.003c	n.d.	n.d.
HES	0.02d	0.02d	0.40a	0.04c	n.d.	0.01e	0.10b	n.d.
L	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.18a
NG	0.03f	0.20b	0.10c	0.10c	0.03f	0.07e	0.08d	0.60a
APG	0.06e	0.20c	0.15c	0.30b	0.09d	0.20c	0.20c	1.91a
KAE	0.10d	0.30b	0.40a	0.40a	0.20c	0.40a	0.40a	0.40a
CHR	n.d.	n.d.	0.03a	0.02a	n.d.	n.d.	n.d.	0.01b
GLN	0.03d	0.04c	0.05b	0.09a	0.02e	0.02e	0.02e	0.03d
Σ	0.6e	2.00d	3.2c	5.1b	0.3f	3.2c	1.5d	16.11a

• Samples (in %): 2A-75% cow's milk + 25% soy beverage; 3A-50% cow's milk + 50% soy beverage; 4A-25% cow's milk + 75% soy beverage; 5A-100% soy beverage; A – L. acidophilus; 2C-75% cow's milk + 25% soy beverage; 3C-50% cow's milk + 50% soy beverage; 4C-25% cow's milk + 75% soy beverage; 5C-100% soy beverage; C - L.casei. GC - Galocatechin; C– Catechin; CoA - p-Coumaric acid; QUE - Quercetin; EA - Ellagic acid; API - Apigenin_7-O-apioglucoside_ (Apiin); SNA - Synapic acid; MYN - Myricetin; DN - Daidzein; HES - Hesperetin; L - Luteolin; NG - Naringenin; AP - Apigenin; GN - Genistein; KAE - Kaempferol; CHR - Chrysin; GLN – Galangin. ^abcDuncan's test confirmed a statistically significant difference between the mean values (± SD) of the content of polyphenolic compounds of different samples at the end of fermentation at the level of significance p < 0.05.

Table 4
Effect of cow's milk and soy beverage in fermented beverages on isoflavone content (daidzein and genistein).

Samples	Genistein (mg/kg)	Daidzein (mg/kg)	Genistein + Daidzein
1A	-	-	-
2A	3.80h	1.79h	5.59
3A	13.70e	5.20e	18.9
4A	11.70f	4.90f	16.6
5A	20.30a	7.80b	28.1
1C	-	-	-
2C	8.20g	3.10g	11.3
3C	16.70d	6.20d	22.9
4C	18.03b	6.30c	24.3
5C	17.70c	11.20a	28.9

• Samples (in %): 2A-75% cow's milk + 25% soy beverage; 3A-50% cow's milk + 50% soy beverage; 4A-25% cow's milk + 75% soy beverage; 5A-100% soy beverage; A – L. acidophilus; 2C-75% cow's milk + 25% soy beverage; 3C-50% cow's milk + 50% soy beverage; 4C-25% cow's milk + 75% soy beverage; 5C-100% soy beverage; C - L.casei. ^abc Duncan's test confirmed a statistically significant difference between the mean values (± SD) of the content of polyphenolic compounds of different samples at the end of fermentation at the level of significance p < 0.05.

According to the results shown in Table 3, it can be seen that quercetin flavonoid is present in all produced fermented samples of different ratios of cow's milk and soy beverage as well as in samples from pure soy beverage, and its content ranged from 0.005 mg/kg (2C) to 0.07 mg/kg (5C). In addition to quercetin, the samples also contained significant amounts of ellagic acid, catechin, and gallocatechin. In addition to these compounds, the flavonoids hesperetin, naringenin, apigenin, kaempferol and galangin were also detected in the samples. Kaempferol was identified in all samples of fermented beverages, and the highest amounts were recorded in samples of pure soy beverages. The presence of flavonoids in different proportions of beverages produced was in variable concentrations, naringenin was present in the range 0.03–0.60 mg/kg, hesperetin 0.01–0.40 mg/kg, and apigenin 0.06–1.91 mg/kg.

Of the isoflavonoid compounds, according to the literature, the most common in soy and soy products are daidzein and genistein. These two isoflavones were analyzed in fermented cow's milk and soy beverages. The daidzein content ranged from 1.79 mg/kg (2A) to 11.20 mg/kg (5C), while the genistein content ranged from 3.80 mg/kg (2A) to 20.30 mg/kg (5A), which is consistent with research by other authors (GolKhoo et al., 2008 ; Chun et al., 2008). Larger amounts of isoflavones were present in samples produced from 100% soy beverage, and the concentration decreased as the proportion of soy beverage in cow’s milk decreased. According to research conducted, higher amounts of isoflavonoid compounds are present in soy compared to their content in soy beverage and soy beverage products. In general, processed products showed lower levels of isoflavones isolated from raw products, due to technological processes such as soaking, heating, and processing, which led to significant losses of isoflavones (Jackson et al., 2002; Huang et al., 2006; Ishihara et al., 2007). Also, the ratio of water and soy, grinding and separation methods, coagulation conditions (Poysa & Woodrow, 2002; Mujić et al., 2011) have an impact on the content of isoflavones as well as other components in processed soy products. Séguin et al. (2004) reported that the content of polyphenolic compounds is significantly influenced by the variety, year, and place of soy cultivation. When it comes to isoflavones, genistein has gained the most attention in isoflavone research because of its potential positive health effects (Šertović et al., 2012). The addition of soy beverage to cow's milk in ratios of 50 or 75% had a large effect on increasing the concentration of flavonoids such as quercetin and kaempferol as well as on increasing the concentration of isoflavones (genistein and daidzein). Analysis of variance showed that there were statistically significant differences in the content of total and individual polyphenolic compounds between samples produced from different ratios of cow's milk and soy beverage as well as the bacteria used at the end of fermentation.

3.3 Analysis of the main components

Analysis of variances in physicochemical parameters showed that there are statistically significant differences between the tested samples of fermented beverages of different ratios of cow's milk and soy beverage. The principal components analysis (PCA) was performed to study the interrelationships between different variables, and in this case, it's the physicochemical parameters. PCA was performed on the results obtained with the produced fermented cow’s milk beverages and soy beverage. The first major component (PC1) accounted for 85.08% of the total data variability, the second major component (PC2) for 7.69%. The value of PCA, ie their mutual projections for the first two components are presented in Figure 1. According to the obtained results of fermented beverages, the first-factor PC1 physicochemical parameters that best correlate are the proportions of Mg, K, S, Fe, Ni, Mn, Cu, Zn, Al, Quercetin (QUE), Daidzein, Genistein, and Kaempferol (KAE), 5C-100% soy milk). Another factor PC2 physicochemical parameters that are positively correlated are the proportions of Ca, Na, and P (2A, 2C - 75% cow's milk + 25% soy beverage; 3A-50% cow's milk + 50% soy beverage).

Figure 1
PCA analysis of the chemical composition of probiotic beverages of cow's milk and soy beverage.

4 Conclusion

Functional probiotic beverages have been successfully produced from different ratios of a mixture of cow's milk and soy beverage with probiotic cultures of L.acidophillus and L.casei. Having in mind the importance of minerals in the diet, it can be concluded that all variants of fermented dairy beverages are a very rich source of calcium, potassium, and sodium. According to the obtained results, fermented beverages produced from different ratios of cow's milk and soy beverage have a significantly more favorable content of analyzed minerals compared to 100% cow's milk samples, which gives the advantage of using soy beverage over cow's milk. By analyzing the content of individual polyphenolic components, it was found that increasing the amount of soy beverage in the mixture increases the number of phenolic compounds present. Quercetin is the most abundant flavonoid present in soy. Based on the obtained results of physicochemical changes in the quality of fermented dairy beverages produced from a mixture of cow's milk and soy beverage, concerning the characteristics of products obtained using only cow's milk, the justification of using soy beverage in the production of fermented dairy beverages can be explained, and to obtain a new functional fermented dairy product with a high content of bioactive components, mineral elements, significant nutritional and organoleptic properties.

• Practical Application: The use of soy beverage in the production of fermented milk beverages with the aim of obtaining a new functional fermented milk product.

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Publication Dates

• Publication in this collection
  14 Mar 2022
• Date of issue
  2022

History

• Received
  05 Sept 2021
• Accepted
  22 Dec 2021