Effect of honeydew honey addition on the water activity and water holding capacity of kefir in the context of its sensory acceptability

The aim of the research was to check how the addition of honeydew honey and various compositions of starter cultures affects the water holding capacity, water activity, color, syneresis and consistency of the obtained kefir in the context of its sensory acceptability. In this research, 2.5% and 5% (w/w) honeydew honey was added to the samples of model kefir (K) and commercial kefir (K13). Kefirs differed by the type of used starter cultures and conditions of production. The addition of honeydew honey to kefir resulted in increased water holding capacity and a reduction in water activity. Honeydew honey kefir was characterized by the following flavor: astringent, fruity, pungent and waxy. As the honey content increased, the taste and waxy flavor became sweeter. In the sensory assessment, the attributes of texture and mouthfeel, creaminess, density and firmness, do not change because of the honey amount or storage time of the samples. The use of different starter cultures in kefir production with the addition of honeydew honey impacted texture parameters, resulting in up to a 4.8-fold increased viscosity index.

honey as a functional additive in fermented milk 16 . In addition, it is used as a sweetener and as a preservative in dairy products 17 . Research also indicates that honey-based kefir beverages are characterized by high antioxidant activity 18 . Furthermore, adequate amounts of honey do not negatively affect bacterial growth; for example, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus and Bifidobacterium bifidum 19 . According to Varga, the addition of 1-5% (w/v) honey had no significant effect on the viability of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus during cold storage of yogurt. Furthermore, they found that addition of approx. 3% (w/v) of honey significantly improves the sensory quality of the finished product 10 . However, when Sert et al. examined the addition of 2%, 4%, and 6% (w/v) of sunflower honey, they showed that its presence in yogurt influences the growth and liveliness of Lactobacillus delbrueckii subsp. bulgaricus during the incubation and storage period of yogurt samples 19 . Coskun and Karabulut Dirican analyzed yogurt with addition of 2%, 4%, and 6% of pine honey, where the number of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus decreased and the number of Streptococcus thermophilus increased compared to the control sample without honey addition. However, the numbers of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus were above the recommended minimum number (≥ 10 6 cfu/g) 17 .
Lactobacillus bacteria are considered to be organisms with high antimicrobial and probiotic potential 20 . Păucean et al. showed that the addition of acacia honey in kefir production at 1%, 2.5%, and 4% (w/v) had no effect on the growth and viability of Lactococcus bacteria. Moreover, the same authors proved that the addition of honey reduces syneresis and increases the consistency of kefir, and no significant effect on pH and lactic acid production was shown 21 . Compared to blossom honeys, honeydew honey possesses potential health-promoting value due to its high bioactive compound content, including phenolics, proteins, and amino acids 22 . The growing market for this honey type significantly contributes to its increased price; however, consumers increasingly appreciate the taste and nutritional value of honeydew honey 23 . According to the literature reports, honey is mainly added to yogurt 10,16,17 . The addition of honey, especially honeydew honey to kefir, is not commonly described in the literature. Therefore, we combined the two products together: kefir and honeydew honey. We decided to take this step also, because most of the research on fortified kefir-based beverages focuses mostly on introducing vegetables as a source of bioactive ingredients. Our observations are confirmed by the review of the literature Aiello et al. The authors also noted the use of plant extracts and essential oils as a source of bioactive molecules in the production of kefir 6 . However, Du and Myracle described the possibilities of using aronia and elderberry in the production of kefir and thus obtaining sensory-acceptable functional food 24 . Similar scientific reports also suggest the use of blueberry and blackberry in the production of fermented beverages 25 .
In view of the above-the high pro-health value of kefir and honey prompts us to research the quality, mainly syneresis, texture and color resulting from the holding of water in the product. Awareness of the combination of kefir and honeydew honey of different densities may result in delamination of the mixture, which in turn may be reflected in the consumer acceptability of such a product. This work focuses on analyzing how the addition of honeydew honey and different compositions of starter cultures shape the sensory acceptability related to water activity, color, syneresis and consistency of the produced kefir because there are still not enough scientific reports on this topic. The presented work will show whether it is possible to create honeydew honey kefir with acceptable properties, which may have importance for further use in public health.

Materials and methods
Model and commercial kefir samples. Two different samples of kefir were examined. The samples did not differ (p > 0.05) in terms of the content of nonfat dry matter (116.0 g/kg), fat (20.0 g/kg), protein (34.0 g/kg), or pH (4.45), which was confirmed in our preliminary research. We performed preliminary studies during the design of the experiment. Kefirs differed by type of used starter cultures and conditions of production. The first sample of model kefir (K) was produced on a pilot plant scale using factory-scale equipment and was made using 6 bacterial strains: Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar diacetylactis, Levilactobacillus brevis, Leuconostoc and yeasts Saccharomyces cerevisiae as starter cultures, which were commercially available from Lyofast MT 032 LV (Sacco, Cadorago, Italy), and added at 0.25 units per 25/L processed milk. Fermentation ran at 26 °C until pH 4.45 was reached. A two-step cooling to 15 °C for a maximum of 15 min was applied, and the product was poured into unit containers of v = 1 L and further cooled to 6 °C. The second sample of commercial kefir (K13) was a commercial product with 13 bacterial strains: Bifidobacterium lactis, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. lactis, Limosilactobacillus fermentum, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar diacetylactis, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Streptococcus thermophilus and yeasts Debaryomyces hansenii (OSM, Koło, Poland). It was the newest product available to consumers in the local market. Additionally, the study involved the possibility of kefir preparation from the dairy industry with honey by consumers at home.
Bacteria and yeasts were enumerated in the model and commercial kefir by plating 500 mL of each diluted sample on appropriate agar media as described by Nambou et al. 26 . Our preliminary research showed that mesophilic LAB in kefir K and K13 were 6.48 and 6.35 log cfu/mL, respectively, and those in yeasts were 4.53 and 4.62 log cfu/mL, respectively. Cell counts of different microorganisms present in model and commercial kefir meet the requirements of Codex Alimentarius 27 . Preliminary research also included the determination of antioxidant potential expressed as the ability of an antioxidant to scavenge stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) according to the method described by Bierzuńska and Cais-Sokolińska 28 . The antioxidant potentials of samples K and K13 were 2435.0 and 2622.0 µmol Trolox/kg, respectively. In addition, there was no effect of sample type, amount of added honeydew honey or storage time on antioxidant potential. www.nature.com/scientificreports/ Honey. Honeydew honey from silver fir (over 78%, the rest is spruce and pine) was produced at an apiary in the Podkarpacie region (Lubaczów district, Poland). Honey was purchased at the market during a local festival directly from beekeepers. The honey came from an apiary with Carniolan honey bees (Apis mellifera). In the preliminary study, the composition and characteristics of honey were determined. The main components of honey were as follows: water 167.0 g/kg, glucose 272.0 g/kg; fructose 337.0 g/kg, sucrose 51.0 g/kg, maltose 21.0 g/kg, trechalose 1.0 g/kg, melecytose 4.0 g/kg, HMF 0.34 mg/kg, minerals 8.0 g/kg, protein 6.0 g/kg, phenolic acid, and p-coumaric acid 26.13 mg/100 g. Properties: electrical conductivity 9.2 × 10 −4 S/cm, pH 4.31, antioxidant activity 1620.34 µmol Trolox/kg, total phenolic compounds 301.22 mg gallic acid/kg, vitamin C 4.3 mg/100 g. Honey was no more than 2 months old and stored in glass jars (v = 250 mL) at room temperature in a dark, dry place.
Experimental design. Kefir was combined with honey no later than the second day after manufacturing (n = 8). Kefir was kept at 6 °C and honey at 18 °C. Mixing was carried out for 10 s using a mechanical stirrer (RPM 20, MRK-12, MPM, Milanówek, Poland). Each sample (v = 1 L) kefir (K and K13) was supplemented with 2.5% and 5% honeydew honey (w/w), and there were 6 samples (including 4 samples with honey). The samples were tested after the completion of mixing (day 0) and at 14 days of cold storage, i.e., at 6 °C ± 0.5 °C.
Determination of water holding capacity. The water holding capacity (WHC) of kefir is defined as its ability to hold all or part of its own water. The WHC of the kefir samples was determined using the centrifugation method 29 . Kefir (30 g) was centrifuged (model 260; MPW MED Instruments, Warsaw, Poland) under relative centrifugal force (RCF) = 10 732 g, 30° (RPM 10 062 g) at 4 °C for 15 min. The supernatant was collected and weighed, and WHC was calculated according to Eq. 1: where W 1 is the weight in grams of the supernatant after centrifugation and W 2 is the weight of kefir in grams.
Spontaneous whey syneresis. The siphon method described by Bierzuńska et al. was employed in the study. A cup of kefir (100 mL) was tilted 45° immediately after being removed from the refrigerator to collect the surface whey; this was siphoned out using a graduated syringe with a needle attached. Siphoning was performed within 10 s to avoid forced leakage of whey from curd 30 .
Water activity. The water activity was measured using an AquaLab Series 4TE instrument (Decagon Color. The instrumental color measurement was based on the CIELab system described by Cais-Sokolińska et al. 32 . The measurement was performed with geometry SPIN using an X-Rite SP-60 camera (Grandville, MI, USA) equipped with a spherical geometry (diffusive) and the measurement chamber with a DRS-811 ceramic insert. The camera was calibrated based on the white and black reference standards SP-62-162 (Grandville, MI, USA). The chrome (C*) (Eq. 2), white index (WI) (Eq. 3), yellowing index (YI) (Eq. 4) were calculated using the following equations: For the calculations, it was assumed that L = 100, a* = 0 and b* = 0.
Sensory analysis. Sensory analysis was conducted via the profiling method 33,34 . Panelists: Thirteen people aged between 20 and 54 were adequately trained individuals prepared to perform sensor examinations 35,36 . Samples were evaluated using 8 cm unstructured line scales anchored with the terms low (denotes an undetectable points parameters) at the left and high (very intense) at the right. Sample temperature was 10-12 °C. The descriptors are listed in Table 1.
Statistical analyses. Verification of statistical hypotheses was achieved using a level of significance of α = 0.05. The influence of the composition and storage time on the samples was evaluated by two-way analysis

Results and discussion
Acidity, activity and mobility of water and water holding capacity in kefir with honeydew honey. Analysis of the physicochemical properties showed no statistically significant differences in acidity in the K and K13 controls, as shown in  www.nature.com/scientificreports/ (K_5.0), and by approx. 10% in commercial kefir with 2.5% honeydew honey (K13_2.5) and commercial kefir with 5.0% honeydew honey (K13_5.0) (p < 0.05). The addition of honey (regardless of the quantity) only to the model kefir did not change the acidity during storage (p > 0.05).In commercial kefir (regardless of the amount of honeydew honey introduced), the acidity decreased by approx. 7% during storage (p < 0.05). Elenany, who analyzed goat milk yogurt with the addition of marjoram honey, found that the increase in acidity of fermented milk may be related to the presence of prebiotic oligosaccharides in honey which may promote the growth and the metabolic activity of lactic acid bacteria 37 . The disadvantage of sensory attractiveness of fermented milk is syneresis, which is a consequence of shrinkage of milk protein gel, which decreases the size of casein aggregates and leads to the separation of whey 38 . Whey separation is affected by various factors (pH, acidity, total solids, hydrocolloid content, etc.). Syneresis is the expulsion of whey from three-dimensional networks, which become visible on the surface, which affects the abbreviation shelf life of fermented milk due to changes in appearance and texture 39 .
The results in Table 2 show that the addition of honey to kefir causes a twofold increase in spontaneous whey syneresis (SWS) in samples K_2.5 and K_5.0 compared to sample K and a fourfold increase in the case of kefir K13_5.0 compared to sample K13 (p < 0.05). However, only a twofold increase in SWS during storage was observed in K13_5.0. It possesses the highest water holding capacity (WHC), with a 21.8% higher value than K. According to Sert et al., this is related to the fructose content of honey, which is capable of binding to water 19 . However, only in the K_2.5 and K_5.0 kefirs were there no statistically significant differences in WHC during storage (p > 0.05). The addition of 5% honey, regardless of the starter culture used, reduced WHC (p < 0.05).
Texture and color of kefir with honeydew honey. Rheological property analysis is important in the determination of the various interactions in kefir samples. Maintaining the proper texture of fermented milk can be challenging in the commercial manufacturing of alternative fermented dairy products 40 . In this study, as shown in Table 3, no statistically significant differences were found in the analysis of firmness, consistency and cohesiveness in kefir produced by Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar diacetylactis, Levilactobacillus brevis, Leuconostoc and yeast Saccharomyces cerevisiae regardless of the additive used (p > 0.05). Kefir produced using 13 strains of bacteria and yeast exhibited greater firmness by 22.4% and 4.8-fold higher viscosity index compared to sample K (p < 0.05). K_2.5 shows increased parameter properties, including firmness, consistency and viscosity index texture, of 6.9%, 7.8% and 26.3%, respectively (p < 0.05). However, no variances were found for K_5 (p > 0.05). There were no statistically significant differences in firmness, consistency or viscosity index during the cold storage of model kefir (K) with the addition of honeydew honey (p > 0.05). The texture of fermented milk is of great importance for the acceptability of the product, therefore it should be stable throughout its storage period 41 . According to Păucean et al., the addition of honey may increase the perceived viscosity of the samples because its addition can promote greater total solids concentrations 21 . However, according to Mohan et al. 's texture parameters, for example, firmness was positively correlated with the total solids 42 . The texture of kefir is a reflection of the ratio of casein to whey protein fractions and the size of casein micelles in the milk being fermented. Casein proteins are found in milk as micelles that form a colloidal solution. Micelles consist of monomers of individual casein fractions linked together by bridges formed by calcium, phosphate and citrate ions. On average, there are approximately 7·10 13 micelles in 1 cm 3 of milk, and their diameter significantly affects the measure of internal friction, that is, viscosity. In cow's milk, the proportion of the α s1 -casein fraction affecting the consistency is 48.5% of the total casein protein (25 g/kg) 43 .
The addition of 5% honey reduced the brightness parameter (L*) by 4.7% and 5.3% compared to the control samples K and K13, respectively, as shown in Table 4 (p < 0.05). In addition, the greatest white index was observed, which was independent of the starter culture used in the production of kefir (p < 0.05). 2.5% honey did not change the chrome C* (p > 0.05). During cold storage, no parameter changes in the brightness L* and white Table 3. Changes in texture parameters in kefir with honeydew honey during storage. K, model kefir; K13, commercial kefir; K_2.5, model kefir with 2.5% honeydew honey; K_5.0, model kefir with 5.0% honeydew honey; K13_2.5, commercial kefir with 2.5% honeydew honey; K13_5.0, commercial kefir with 5.0% honeydew honey; d, day; SEM, standard error of the mean (n = 8). a-e, A-B Different lowercase letters in the superscript in rows and capital letters in columns for each parameter indicate statistically significant differences at the level α = 0.05.  42 . Color changes are possible at all stages of milk processing, e.g., the Maillard reaction during heating 44 . The color change is also related to the fermentation process. Therefore, instrumental color measurement becomes important 45 Fig. 2. The results show that as honey content increases, kefir has more fruit and astringent flavors. The difference between the addition of 2.5% and 5.0% of honey is not significant. However, compared to kefir without the addition of honeydew honey, the flavor pungent is 3 times more perceptible. Regardless of the starter culture used, the addition of 2.5% honeydew honey had no influence on flavor, including invigorating and kefir-like flavors, or attributes of texture and mouthfeel: prickling. The addition of 5% honeydew honey to kefir can be characterized by attributes of taste (sweet) and flavor (waxy). It is presumed that the sweet and waxy flavor may have the greatest impact on the assessment of their acceptability by consumers. Samples K_5.0 and K13_5.0 have sweet tastes with scores of 6.8 and 7.7 on a scale of 1-9, respectively, making the samples more like honey than kefir. Additionally, high marks are obtained for the waxy flavor of the samples K_5.0 and K13_5.0 (6.5 and 7.7 score, respectively). Hence, the supposition that the addition of 5% may be too high and that the addition of honey to kefir in the amount of 2.5% is more beneficial with consumers in various age groups. According to Larosa et. al use of honey for kefir improves acceptance in appearance and aroma 46 . Păucean et al. showed that the optimal addition of honey to kefir was 2.5% (w/v), and the introduction of 4% honey (w/v) made the kefir too sweet. Additionally, they found that improvement of syneresis, consistency, taste and flavor can be obtained with 2.5% honey, and the production of kefir with the addition of honey may be an alternative for desired taste and nutrition for new fermented dairy beverages 21 . However, Mohan et al. showed that the introduction of 5% (w/v) Manuka honey to yogurt can enhance both the functional health value and consumer acceptance 42 .

Conclusions
The addition of honeydew honey to fermented milk causes greater WHC and reduces water activity, regardless of the type of starter culture used. The use of a different starter culture in kefir production with the addition of honeydew honey has an impact on the texture parameters, resulting in up to a 4.89-fold increase in the viscosity index. Measuring the parameters of texture, color, and syneresis of fermented milk with honey is important due to the development of its quality and associated consumer acceptability. Implementing honeydew honey into kefir is a novel food example. At the same time, the entire technological protocol of kefir with honey was developed. As a result of combining the well-known kefir and honey, it is possible to obtain a product with completely new properties and enriched value, which may be a response to the new needs of the consumers. This is important due to the growing awareness of the consumer about the relationship between diet and health. Table 4. Change assessment of the color of kefir with honeydew honey during storage. K, model kefir; K13, commercial kefir; K_2.5, model kefir with 2.5% honeydew honey; K_5.0, model kefir with 5.0% honeydew honey; K13_2.5, commercial kefir with 2.5% honeydew honey; K13_5.0, commercial kefir with 5.0% honeydew honey; d, day; L*, brightness; WI, white index; C*, chrome; YI, yellowing index; SEM, standard error of the mean (n = 8). a-e, A-B Different lowercase letters in the superscript in rows and capital letters in columns for each parameter indicate statistically significant differences at the level α = 0.05.