QUALITY OF KEFIR PRODUCED USING ACTIVEFLORA PROBIOTIC

Anna Mituniewicz-Małek¹, Izabela Dmytrów¹, Małgorzata Jasińska^2
1 Department of Dairy Technology and Food Storage, West Pomeranian University of Technology, Szczecin, Poland
² Department of Dairy Technology and Food Storage, Faculty of Food Sciences and Fisheries, West Pomeranian University of Technology, Szczecin, Poland

ABSTRACT

In this study we examined the qualitative characteristics of cow milk kefir, produced using a lyophilized, probiotic ActiveFlora (AF) preparation, manufactured by Bionat. The control kefir was produced using a lyophilized DA kefir culture, the best among the kefir cultures manufactured by Danisco Poland (DA, DC, DG, DT and DL). DA inoculum included the microflora of kefir grains, and also Lactobacillus ssp., Lactobacillus ssp., Leuconostoc ssp., Lactococcus lactis ssp. lactis and kefir yeast. The AF preparation, apart from the Lactobacillus acidophilus-NCFM, contained the natural microflora of Caucausian kefir grains, and Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris oraz Lactococcus lactis ssp. lactis var. diacetylactis. The experimental kefirs were subjected to physiochemical analysis (titratable acidity, pH, acetaldehyde and ethanol concentrations), rheological analysis (hardness and viscosity), and an organoleptic test by a team of researchers. Samples were collected after 1, 3, 7 and 14 days of cool storage (5±1°C).
The results of this study show that kefir produced using ActiveFlora (AF), similarly to DA control kefir, had very good and good organoleptic characteristics. During the first 3 days of cool storage, its scent and flavour were slightly worse than that observed in DA culture kefir, but after 3 days they were slightly better than in DA kefir. Acidity (both titratable and pH) and rheological properties (viscosity and hardness) were similar in both kefirs during the entire period of storage. Greater differences between the examined kefirs were observed in the concentrations of gustatory-olfactory compounds; during two weeks of cool storage, it was the DA kefir that usually had a higher concentration of acetaldehyde and ethanol.

Key words: kefir, acidity, acetaldehyde, ethanol, hardness, viscosity.

INTRODUCTION

One of the oldest and especially valuable fermented milk drinks is kefir, produced in many centuries using kefir grains, formed spontaneously in natural conditions of Caucasus. According to different sources, kefir grains are a unique symbiotical team of microorganisms behaving as one organism, identified by a dozen or so to tens of groups of microbes, including 65-80% lactic acid bacteria, 8-23% streptococci, 2-12% yeast leaven [1,30,41]. Numerous studies showed that microbes constituting kefir grains and their metabolites (mostly lactic acid and ethyl alcohol) play a very important role, both a physiological and medical point of view. According to Fesnak et al. [12], lactic acid introduced to the digestive tract, in a quantity as low as 1% of the intaken liquid, promotes the secretion of digestive juices in the stomach and promotes the activity of intestines. Moreover, it inhibits or destroys injurious and even pathogenic bacteria (Staphylococci, Salmonellae) which limits the processes of putrefaction and consequently improves appetite. The alcohol slightly accelerates the circulation of blood in the human body, and consequently increases the supply of nutrients [32]. Moreover, according to literature on the subject [13,48] the microflora of kefir grains synthesize B group vitamins and partially digest protein and milk-sugar, making them more assimilable for the body. At present, however, because of the troublesome technology of kefir production using kefir grains [11,29], and due to a need to standardize sensory, nutritional, dietetic and biological values, this traditional method is being abandoned. The industrial production of kefir increasingly often applies easy-to-use deep frozen or lyophilized kefir inoculum, and also other preparations with the properties of living kefir grains, which maintain their healthful values but also lower the labour intensity during the inoculum preparation and eliminate the possibility of undesirable microflora, thus improving the quality of the ready product and lengthening its life [42].

In connection with the aforementioned aim of this research, we analyzed selected qualitative characteristics of kefir obtained using ActiveFlora preparation which contained, among other things, grains of Caucasian kefir, during cool storage.

MATERIAL AND METHOD

This study was carried out using kefir produced under laboratory conditions, using a thermostat method at the Dairy Technology Department at the Agricultural Academy in Szczecin, Poland. UHT milk, purchased from retail stores, was used as raw material. The experiment used two batches of kefir. The kefir DA (culture DA), the best from the analyzed groups of kefir cultures produced by Danisco Poland (DA, DC, DG, DT and DL), was produced in the first batch. The second batch was inoculated by ActiveFlora (AF) formulation by Bionat. The culture DA, consisted of kefir grain microflora, and also Lactobacillus ssp., Leuconostoc ssp., Lactococcus lactis ssp. lactis and kefir yeast leaven. The formulation AF, apart from probiotic Lactobacillus acidophilus-NCFM, contained natural microflora of Caucasian kefir grains and also Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis var. diacetylactis. The composition and activity of the microflora used in the production of samples is presented in Table 1.

Experimental kefir was produced using the following method.
UHT milk for kefir production was heated to 22°C per technological instruction [47], and divided into two equal portions. Then each batch of the base milk was inoculated with 5% addition of inoculum obtained from DA or AF culture. Then the material was split (50cc each) to individual containers, incubated (22°C/12-14h), and then subjected to maturation (8°C/12-14 h). The ready kefir was then transferred to a cooling chamber at 5°C (±1°C) and kept for two weeks. Samples were collected randomly after 1, 3, 7 and 14 days of cool storage, 1dm³ altogether (twenty 50 cm³ containers of kefir ) from each batch.

The general protein concentraion in the base milk were determined using Walker's method [50], fat content was determined using Gerber's method [50], dry matter according to Fleichmann [50], acid content using a Soxhlet-Henkel titratable method [4] and pH using a pH meter [50].

In the fermented samples the following parameters were determined: titratable acidity using a Soxhlet-Henkel method [5], pH using a pH meter [50], the concentration of acetaldehyde with a diffusive method using hydrazone hydrochloride (3-methyl-2-benzothiazolinonhydrazon) in Conway cells [23], level of ethyl alcohol using the titratable method after ethyl oxidation to acetic acid [5]. The experimental kefirs underwent also a rheological analysis, which was based on the evaluation of their hardness and viscosity. First analysis was made by means of double compression test (TPA) using a texture analyzer TA.XT Plus of Stable Micro System manufacture. The samples were penetrated with an aluminium cylinder with a diameter 20 mm to a depth of 25 mm and a speed of 5 mm·s^-1 and force of 1G [10]. Viscosity  was determined using a rotational viscometer Rheotest 2-50Hz-Type RV2 with a controlled cutting speed in the S/S2 coaxial cylinder system. Additionally, samples were subjected to organoleptic assessment by a 5-person panel trained in carrying kefir sensory analysis, using a 5-point scale, extended by half-marks. The examination was carried out at a special laboratory, free from outside scents, in compliance with specific recommendations [22], considering appearance, taste, aroma and consistency.

All the determinations, carried out within the range of physicochemical and rheological analyses, were performed with 3 repetitions, and the obtained results were subjected to statistical analysis. The significance of differences between the physicochemical and rheological coefficients was estimated using Student's-t test or Cochran-Cox test using Microsoft Excel 2000 Software package. All test were made at significance level α=0.05.

RESULTS AND DISCUSSION

The base milk used in kefir production contained 11% dry matter, including 3.6% proteins and 3% fat, titratable acidity and active acidity (pH) were 6.6 °SH and 6.3 pH, respectively. Following are the results of physicochemical and rheological analyses of the kefir obtained using ActiveFlora in respect to the control kefir.

Titratable acidity of experimental kefirs ranged from 32.1 °SH to 35.6 °SH, and the obtained results showed that this parameter was more influenced by the duration of storage than the type of applied culture (Fig. 1). Accordingly, during the two weeks of the experiment, the greatest oscillations of the titratable acidity were observed after the 7^th day of cool storage, when in both analyzed kefirs the value of the examined coefficient increased by approx. 2°SH on average, compared to the value recorded after the 3^rd day of the experiment. However, it must be noticed that both during the first (1^st day) and in the last period of the experiment (14^th day), kefir with the DA culture had a higher acidity than the kefir with the AF preparation. The performed statistical analysis showed that differences in the acidity of kefirs were statistically significant only after the 1st day of storage (Table 2).

The probiotic AF preparation, applied in the production of the experimental kefir did not have a significant influence on the level of active acidity (pH) (Table 2). Within two weeks of the experiment, pH ranged from 4.14 to 4.46 pH, and similarly to titratable acidity, depended more on the time of cool storage (Fig. 2). Accordingly, the oscillations of the analyzed coefficient in both types of kefir were observed after 7 days. In the DA kefir pH decreased by 0.32 units on average, and in AF kefir by 0.27 units, compared with the initial value (after 1 day). In the last stage of storage (after 14 days), both types of kefir showed a slight increase in pH – in DA kefir acidity was higher by 0.12 units, and AF kefir by 0.13 units, compared to values after the first week of the storage (after 7 days). A similar plot of acidity (titratable and pH) in fermented samples made from cow milk during cool storage was observed by Pieczonka and Pasionek [38], Cais-Sokolińska and Pikul [6], as well as by Bonczar and Wszołek [4] in kefirs and yoghurts from sheep milk, explaining that the observed results resulted from the fermentative activity of microbes included in cultures, which at a temperature of 4°C still dissolve lactose, although considerably slower than at temperatures optimal for the proliferation of lactic fermentation bacteria. Moreover, gradual decrease of pH in the kefir, in comparable period (up to 8 days), was shown by Garcia Fontan et al. [14]. At the same time slightly different values of titratable acidity and pH in kefir were observed  by Tratnik et al. [46].

The experimental kefirs were analyzed with regard to the concentration of gustatory-aromatic compounds (acetaldehyde and ethyl alcohol) whose levels depended on the type of the applied inoculum. In the case of acetaldehyde, within two weeks of the research, it was the DA kefir that in general had a considerably greater concentration, ranging from 0.148 to 0.529 mg·dm^-3 (Fig. 3). The greatest differences in its level were observed after the 3^rd and 7^th days of cool storage, and were statistically significant (Table 2). In both examined types of kefir, acetaldehyde concentrations changed in different ways over the duration of storage. During the first week, in the DA product it increased, and in AF kefir it decreased, compared with the 1st day of storage. In the DA product, after 7 days, the concentration of acetaldehyde increased more than two times, and in the AF kefir it decreased by approx. 99%. During the last week (after 14 days), a decrease was observed in the DA kefir, and in the AF kefir a significant increase was recorded, compared to the concentration after 7 days of storage.

Ethyl alcohol concentration (Fig. 4), during the two weeks of the experiment, also increased in DA kefir (up to 0.0065–0.0155%). As early as after 1 day of storage, the greatest ethanol concentration (0.0193%) was observed in AF kefir and was higher by approx. 30% compared to DA kefir, and such differences were also observed after 3 and 14 days (Table 2), all statistically significant differences. Regardless of the type of kefir, the concentration of ethyl alcohol in each day of cool storage was very low and gradually decreased over time. The greatest decrease in both types of kefirs was observed after the first week of cool storage. After 7 days, in the DA kefir sample, the concentration of ethyl alcohol was lower by approx. 51%, and in the AF kefir sample by approx. 83%, compared to values after the 1^st day.

The specific taste and aroma of fermented products depended on the accumulation of metabolites during the joint growth of microorganisms from the applied cultures. One of the components of their aroma is acetaldehyde. In spite of the fact that according to literature the mentioned compound is a basic component of yoghurt's aroma [24,35,41] but not kefir's aroma [20,44], in products obtained using mesophilic inoculum, a certain amount of acetaldehyde is desirable, as it polishes by scraping taste of diacetyl. Another basic gustatory-aromatic compound, that is characteristic for fermented milk-drinks, such as kefir, is ethyl alcohol, produced mostly by lactose-fermenting yeast, the concentration of which can even reach the level of 1–2% according to literature [21,44]. This study showed the concentration of the analyzed gustatory-aromatic compounds (acetaldehyde and ethyl alcohol) in the examined  kefir depended on the type of the used inoculum, and also on the storage duration. One may suppose that the differences in the level of acetaldehyde between experimental samples resulted from a different qualitative and quantitative composition of microflora in inocula used in production, in other words from the diverse activity of threonine aldolase produced by each bacteria. According to available literature [24,25,28] microflora used in the production of acidophilic milk differ with respect of the activity of this enzyme which participates in the formation of acetaldehyde from nitric compounds. Additionally, Libudzisz [26] ascertained that the dynamics of formation of gustatory-aromatic compounds in fermented milk depend not only on the species but also on the strain of bacteria. Significant influence of inoculation on formation of aromatic components in acidophilic milk was also staded by Beshkova et al. [3]. One needs to stress that the concentration of acetaldehyde in experimental kefirs was distinctly lower than in yoghurt with a correct aroma (10mg·dm^-3), but in general in accordance with literature [20,43], lactic fermentation bacteria can produce 0.1–10.0 mg·dm^-3 of this compound. Similar results were obtained by Ott et al. [36]. The low level of acetaldehyde in cow milk kefir was also observed by Molska et al. [32] and Magdalińska [27]. In this study, experimental kefirs had a low concentration of ethyl alcohol (0.0024–0.0199%), which could have resulted from the weak development of yeast in leaven used in the kefir production, and also from the fact that experimental samples were obtained using lyophilized kefir cultures and not using traditional kefir grains. Literature on the subject [15,37] shows that 1 day-old kefir produced directly on kefir grains may contain up to 0.2%, and 3-day kefir even 0.8–1% of ethyl alcohol , while kefir made with leaven usually contains below 0.1% ethyl alcohol. Significant differences in ethanol contents between traditional (1.72%) and modified kefir (0.3%) were also stated by Muir et al. [34]. A greater ethyl alcohol concentration in cow milk kefirs, was observed by Guzel-Seydim et al. [16]. Similarly Tratnik et al. [46], who added that according to many authors the ethanol concentration in kefir varies, and can range from as low as 0.002% to 0.03%. Moreover, considering the composition of microflora cultures used for the production of the experimental kefir in this study (at least those declared by manufacturers), one may conclude that the significant differences in the concentration of ethyl alcohol between DA and AF kefir samples resulted from differences in their microflora composition. The same conclusions were reached by Jasińska et al., [17,18], at the same time observing a low concentration of the examined compound in the analyzed group of kefirs, as well as jak również Beshkova et al. [2].

The experimental kefirs in this study were analyzed with respect to their hardness and viscosity (Fig. 5 and 6) During two weeks of cool storage the hardness of the examined products ranged from 19.2015 to 23.825 G, and a statistical analysis showed no significant differences in the level of the analyzed parameter between the DA kefir samples and AF ones (Table 2). However, in the initial period of storage, higher hardness was observed in AF kefir. After 1 and 3 days, its hardness was approx. 8% and 17% higher respectively compared to DA kefir samples.

The lack of significant differences between the products was also observed for viscosity (Table 2) which over the three weeks of cool storage ranged from 20.782 to 28.748 mPa·s. However, it is interesting that DA kefir, except at day 7, had a higher viscosity than AF kefir. The greatest difference was observed after 3 and 21 days of cool storage, where in the first case (after 3 days) viscosity of DA kefir was approx. 9% higher compared to AF kefir, and in the latter (after 14 days) by approx. 12%. The value of the examined parameter depended on the duration of storage; the lowest viscosity was observed after 1 day, and greatest after 3 days of cool storage.

To sum up, the obtained results of hardness and viscosity showed they were not significantly influenced by the composition of DA and AF microfloras used in the production of the experimental kefir. They depended more on the duration of storage, and the greatest oscillations in hardness and viscosity were observed in the first week of cool storage. The plot of hardness, in relation to the applied starter culture, was also examined by Torre et al. [45] and Mituniewicz-Małek et al. [31] in cow milk yoghurt and goat milk yoghurt respectively. The analyzed parameter was influenced by the composition of microflora of the culture applied in their production. The lack of consistency with our results is associated with the fact that the examined biological material in this study was different. Moreover, the quoted publications compared several inoculum, which guaranteed the appearance of differences, as opposed to this research which  compared only two variants. Nevertheless, Torre et al. [45], during the storage of each experimental kefir, observed the greatest differences in the level of hardness in the first days of the experiment (until the 8th day), as was observed in this research. Also many other authors [2,4,9,19,39,46] observed the influence of storage duration on the viscosity of fermented milk-drinks (kefir and yoghurt), obtained from the milks of cows, goats and sheep. Additionally Beshkova et al. [2] observed difference in viscosity between  kefirs differentiated with respect to selection of inoculation.

The organoleptic assessment in this study showed that AF kefir and control DA kefir, had very good and good qualitative characteristics over the duration of cool storage (Fig. 7). However, as early as after the 3^rd day of storage, both types of kefirs were observed to have a slight whey outflow (syneresis), which remained until the end of the experiment in the case of AF kefir . The occurrence of syneresis after 3 days, negatively influenced the consistency of the experimental  kefirs, which was far less compact than in the remaining experimental cycles (after 1, 7 and 14 days). Whey outflow as early as after 3 days of cool storage was also observed by Magdalińska [27] in the group of kefirs bought in the area of Szczecin, Poland. The literature reports that occurrence of synaeresis in fermented milk-drinks is caused by several factors; the chemical composition of raw material, use of additives responsible for the increase in dry matter in milk, the selection of inoculum and the applied technology. Also heat-treatment is crucial here, and more exactly, appropriate parameters of pasteurization (temperature and duration) which guarantee the proper denaturation of whey proteins (by 80%) which results in better water binding, improved coagulation and most importantly an increases in the resistance to synaeresis [7,51]. According to Ziajka and Dzwolak [49], the proper quality of fermented milk depends on pasteurization at a temperature of 85–95°C for 5–30 minutes. It is significant because the influence of heat-treatment on the improvement in coagulation and viscosity of the yoghurt is correlated with the degree of denaturation of lactoglobulin-β which, interacting with casein, significantly influences the yoghurt's texture [8]. In conformity with literature, the peak degree of interaction takes place at 85°C for 30 minutes. Heating milk below 85°C and above 120°C causes a distinct decrease in the clot consistency [52]. However, Mottar et al. [33], showed that the consistency of yoghurt does not unequivocally depend on the degree of  whey protein denaturation, all the more that in spite of such denaturation of these proteins in UHT milk and in milk heated for 10 minutes in 90°C, yoghurt obtained from UHT milk was characterized with lower viscosity and consistency. Based on organoleptic assessment, it was also observed that until the 3^rd day of cool storage DA kefir had a little better taste and scent, and after the 3^rd day AF kefir was better. Considering the fact that samples were obtained using the same raw material, it is very probable that organoleptic differences depended on the type of inoculum, and more precisely their microflora.

CONCLUSIONS

To sum up, this research did not show expected organoleptic differences and selected physicochemical and rheological coefficients (especially ethyl alcohol) between kefir produced using ActiveFlora and control kefir. One may suppose that the activity of Caucasian natural kefir grains in the examined probiotic preparation is not significant. Nevertheless, in consideration of the fact that the applied probiotic ActiveFlora assured, in spite of all, proper quality of the product, and additionally as a source of precious microflora, it is recommended for kefir production, with particular reference to production in household production.

1. AktiveFlora priobiotic, during the whole period of cool storage, provided good and even very good organoleptic properties of the ready product.

2. The acidity (titratable and active) and rheological properties (viscosity and hardness) of kefir obtained using ActiveFlora were close to the values in the control samples.

3. ActiveFlora did not have a significant influence on the concentrations of gustatory-aromatic compounds (acetaldehyde and ethyl alcohol), although in general their concentrations were higher than the kefir control.

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Accepted for print: 1.07.2009

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