LWT - Food Science and Technology 154 (2022) 112728

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LWT
journal homepage: www.elsevier.com/locate/lwt

A new functional kefir fermented beverage obtained from fruit and

vegetable juice: Development and characterization
Jorge Luís Paredes , María Luisa Escudero-Gilete *, Isabel María Vicario
Food Colour and Quality Laboratory, Facultad de Farmacia, Universidad de Sevilla, 41012, Sevilla, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: A juice containing a mixture of fruits and vegetables (70% apple, 9% strawberry, 12% carrot and beet 9%) was
Kefir evaluated as a potential substrate for the production of a novel probiotic beverage made with kefir grains. The
Sensorial analysis effects of the kefir grains amount (1–4%, w/v) and fermentation time (12, 24 and 48 h) on the beverage
Antioxidant
composition, sensory qualities and colour were investigated. The results indicated that the amount of kefir grains
Probiotic
Lactic acid bacteria
have a significant effect on the content of the organic acids (lactic, acetic, citric, succinic, and malic acid), CO2
production, acidity, and viscosity and colour parameters (lightness (L*), hue (hab) and Chroma (C*ab)).
Fermentation time also significantly affected all the parameters analyzed in the samples. The most suitable
conditions to achieve the highest overall acceptability for the fermented beverage based on a mix of fruits and
vegetable juice was: 2% (w/v) kefir inoculum during 24 h of fermentation time.

1. Introduction Although dairy products are the best substrate for probiotics, there
are some drawbacks related to milk composition like hypersensitivities
Covid_19 pandemic has increased the consumers’ interest in func­ (allergies or intolerance). In Europe, an average prevalence of milk
tional foods that provide health benefits related to immune system and protein allergy of 6–8% in children and 2% in adults is estimated
stress, thereby driving the growth of the functional food market (The (Álvarez Berciano & Álvarez Caro, 2008) while lactose intolerances
Business Research Company, 2021). Among these products are pro­ affect one-third of the world population. Moreover, it is estimated that
biotics which exert a beneficial effect on the intestinal functions and 600 million people in the world are vegetarian while 2%, 4% and 1% of
may be able to prevent several diseases due to the living microorganisms the US, Swedish and German population are vegans. The vegetarian and
(De Las Cagigas & Blanco, 2002). Kefir is an ancient fermented beverage vegan product industries are growing around 10% per year (Choudhary
associated with longevity in the Caucasus (Cevikbas et al., 1994), to & Jadoun, 2014). These are some of the reasons why fermented
which beneficial health effects such as reduction of symptoms of lactose non-dairy beverages have begun to play an important role in the diet of
intolerance, stimulation of the immune system, cholesterol-lowering, consumers who are hypersensitive to milk proteins (Álvarez Berciano &
anti-mutagenic and anti-carcinogenic properties are attributed Álvarez Caro, 2008), lactose intolerant (FEN, 2013), vegetarians and
(Güzel-Seydim, Seydim, Greene, & Taş, 2006). vegans (Choudhary & Jadoun, 2014) also new studies on lactic acid
Kefir is a symbiotic medium of microorganisms characterized by fermentation of fruit juices are being conducted to bio-enriched in se­
presenting a mass composed of proteins, lipids and a soluble poly­ lenium (Crespo et al., 2021; Gaglio et al., 2021).
saccharide called kefiran where its microbiota, a spectrum of lactic acid The juices of fruits and vegetables are rich in sugars that can be a
bacteria, yeasts, and acetic bacteria, is found. The microbiota of kefir fermentative substrate for kefir (Alves et al., 2021; Bueno et al., 2021).
grains is formed by sugar-fermenting microorganism that produces Many studies have linked consumption of fruits and vegetables with a
lactic acid, alcohol, CO2, B-complex vitamins, and other organic acids in reduction of the risk for several chronic diseases, such as cancer, car­
their metabolism. The overall metabolic capacity of this consorcium is diovascular diseases, cataracts, or immune dysfunction (Baines & Seal,
the most important characteristic of stable water Kefirs (Gulitz, Stadie, 2012; Garcia, Guerin, Souidi, & Remize, 2020). These natural protective
Wenning, Ehrmann, & Vogel, 2011; Stadie, Gulitz, Ehrmann, & Vogel, effects have been attributed to the antioxidant potential of several
2013). components, such as carotenoids, betalains, vitamins, polyphenols, and

* Corresponding author.
E-mail address: [email protected] (M.L. Escudero-Gilete).

https://doi.org/10.1016/j.lwt.2021.112728
Received 14 June 2021; Received in revised form 8 October 2021; Accepted 27 October 2021
Available online 2 November 2021
0023-6438/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
J.L. Paredes et al. LWT 154 (2022) 112728

other phytochemicals. Some of these compounds, including betaines or Carbon dioxide (g/100 mL) was indirectly estimated by measuring
carotenoids, have health claims authorized under Article 13(1)-Regu­ the weight loss before and after the fermentation (Zilio, Tosi, Lombardi,
lation (EC) No-1924/2006 by the European Food Safety Authority & Delfini, 2004).
(EFSA). Thus, fermented vegetables and fruit juices are a promising The content of lactic, acetic, citric, malic and succinic acids was
approach for therapeutic foods (Noğay, 2019). Currently, the develop­ determined by an HPLC method according to Zaky, Pensupa,
ment of functional beverages based on fruit juices with probiotics has Andrade-Eiroa, Tucker, and Du (2017) with modifications. Analyses
increased due to their health benefits and good acceptability by con­ were carried out in an Agilent 1100 chromatograph (Agilent Technol­
sumers of all ages (Tesfaye, Suarez-Lepe, Loira, Palomero, & Morata, ogy, Palo Alto, CA, USA) equipped with a diode-array detector, which
2019). was set to scan from 200 to 770 nm. The analysis conditions were:
The work aimed to formulate a kefir beverage obtained by fermen­ separation column Hi-plex-H (8 μm, 300 × 7.7 mm), Ta = 30 ◦ C, mobile
tation of commercial fruit and vegetable juice (Veggie) under different phase 5 mM sulphuric acid and 0.8 mL/min flow rate, injection volume
conditions, evaluate the influence of the fermentation conditions on the = 50 μL.
final composition and some bioactive components and finally evaluate Organic acids were identified by their retention time, UV–vis spectra,
its acceptance. and comparison with external standards. They were quantified by
external calibration with calibration curves constructed with external
2. Materials and methods standards.
Total phenolic content was determined using Folin-Ciocalteu assay
2.1. Samples (Singleton & Rossi, 1965). The absorbance was read at 765 nm with a
Hewlett-PackardUV–vis HP8453 spectrophotometer (Palo Alto, CA,
The juice selected was an industrial product to assure a standard USA). Gallic acid was employed as a calibration standard and results
quality and minimize variations. A commercial juice from the same lot were expressed as gallic acid equivalents (mg GAE/L).
(Lot-120738) of Veggie brand, made of apple extract (70%), carrot The antioxidant activity was analyzed according to ABTS/persulphate
(12%), beetroot (9%), and strawberry (9%), was used as base beverage. assay. The ABTS•+ radical was produced by the oxidation of 7 mM ABTS
This juice was selected because of the beetroot in the composition, with potassium persulphate (2.45 mM) in water and allowed to stand in
which, besides an attractive red colour, contains betaine a compound the dark at room temperature for 16 h before use. The ABTS•+ solution
with a health claim authorized under Article 13(1)-Regulation (EC) was diluted with phosphate-buffered saline (PBS) at pH 7.4 to give an
No-1924/2006 ′′ contributes to the normal metabolism of homocysteine” absorbance of 0.7 ± 0.02 at 734 nm. 50 μL of sample was mixed with 2
(Regulation (EU) 432/2012). mL of ABTS•+ diluted solution, vortexed for 30s, and the absorbance
Kefir grains were obtained from a donor. The samples of Kefir grains measured at 734 nm after 4 min of reaction at 30 ◦ C.
were preserved in sterilized milk, renewed daily for two months. These The results were expressed as Trolox-equivalent antioxidant capacity
grains were washed with sterile distilled water and subsequently used to (TEAC; μmols of Trolox with the same antioxidant capacity as 1 L of the
inoculate the commercial juice. studied sample) by interpolating the absorbance on a Trolox calibration
Based on previous studies by Corona et al. (2016) and Sabokbar, curve (30–1000 μM).
Moosavi-Nasab, and Khodaiyan (2015), two different variables were Colour was measured by diffuse reflectance with a spectrophotom­
considered: the kefir grains concentration and the fermentation time: eter CM-5 (Konica Minolta Sensing Americas, Inc., NY). Each sample
four 1%, 2%, 3% and 4% w/v and three levels: 12, 24 and 48 h were was placed in a tube cell of optical glass (CR-A506 Tube Cell Ø60/60 mm
considered respectively 12 different samples were prepared (Table 1). depth). The Illuminant D65 and the 10◦ observer were considered as
The whole experiment was done in triplicate under the same conditions. references. The colour parameters corresponding to the uniform colour
Fermentation temperature was controlled at 26 ◦ C. The fermented space CIELAB (L*, a*, b*, C*ab and hab) (CIE, 1986) were obtained
beverages were sampled to evaluate the physicochemical parameters. directly from the apparatus. Lightness (L*) oscillates between 0 (black)
The samples for the sensory evaluation test were kept under refrigera­ and 100 (white). The coordinate a* take positive values for reddish
tion (4 ◦ C) and in a modified atmosphere (with nitrogen addition) to colours and negative values for greenish ones, and b* takes positive
prevent changes in the organoleptic properties. Fermented beverages values for yellowish colours and negative values for bluish ones. Addi­
were analyzed in triplicate using the commercial juice as the control tionally, two psychological parameters, hue (hab) and chroma (Cab*) are
sample. defined, which are related to a* and b* as follows: C*ab = [(a*)2 +
(b*)2]1/2; hab = arctan (b*/a*). C*ab is regarded as the quantitative
2.2. Physicochemical determinations attribute of colourfulness and enables to determine for each hue its
degree of difference relative to grey colour with the same lightness. Hue
pH, total titratable acidity and soluble solid content were performed angle (hab) takes values from 0◦ to 360◦ and is the qualitative attribute
according to the methodology proposed by the AOAC (2000). that allows any colour to be described as bluish, reddish, etc. This
Viscosity was calculated as per ASTM standard methods D 445 and D parameter allows any colour to be differentiated from a grey one with
2515 using a Cannon-Fenske viscometer at 25 ◦ C. The absolute viscosity the same lightness. The colour differences (ΔE*ab) between two colours
(η) was calculated as η = ctd, where c is the constant: 0,0084295 (at in the CIELAB space are calculated as the Euclidean distance between
50 ◦ C), t (s) is the efflux time, and d (g/mL) is the sample density. their locations in the three-dimensional space defined by L*, a* and b*:
Density (g/mL). 25 mL of sample was weighed, and weight was ΔE*ab = [(ΔL*)2 + (Δa*)2 + (Δb*)2]1/2
divided by volume.
2.3. Sensory evaluation
Table 1
Sample coding according to fermentation time and % of Kefir added. The beverages were subjected to sensory evaluation. 10 mL aliquots
of samples were served, in random order, in clear cups (30 mL volume)
Kefir concentration (% w/v) Fermentation time (hours)
covered with Petri dishes and marked with three-digit random numbers.
12 24 48 A glass of water was offered to the judges between samples to rinse their
1% 1K12H 1K24H 1K48H mouths. The assessments were carried out in individual booths (70 × 70
2% 2K12H 2K24H 2K48H × 55 cm) at room temperature and under white light.
3% 3K12H 3K24H 3K48H Two sensorial analyses were carried out to select the more preferred
4% 4K12H 4K24H 4K48H
sample (Fig. 1). Either a trained or a consumer panel was used

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J.L. Paredes et al. LWT 154 (2022) 112728

Fig. 1. Flow diagram of the sensory analyses leading to the more preferred beverage.

depending on the purpose of the analysis: variance (ANOVA), and statistically significant differences (p < 0.05)
were determined using the Tukey multiple comparison test (Norman &
- The first sensory sessions were aimed at assessing the effect of Streiner, 1996).
fermentation time and kefir concentration on the sensory properties Friedman’s test (analysis of variance by ranks) was carried out when
of the beverages and selecting the 3 most preferred products. A the variables were measured in terms of categories. As recommended by
comprehensively trained 15-judge panel (9 women and 6 men, the ISO 8587, the Friedman test (Friedman, 1937) is for comparing three
23–55 years old) with long expertise in the sensory analysis of food or more related samples and makes no assumptions about the underlying
was recruited among academic staff. Panellists were given 3 to 4 data distribution. The statistic of Friedman test for each sample was
samples in random order in 7 different sessions. The effect of the compared to critical values calculated according to Hollander and Wolfe
fermentation time (4 sessions, 3 samples/session) and percentage of (1973). When according to the Friedman’s test statistically significant
inoculation of kefir on the juice (3 sessions, 4 samples/session) were differences among samples were found, the pairs of differing samples
considered. Rank-order test and preference test were the two pro­ were identified using an analogue of Fisher’s least significant difference
cedures used in each session. The panellist rank-ordered the samples (Fisher, 1922).
according to sweetness, acidity, and alcohol; in addition, they had to Consumer data first underwent normality testing (Shapiro-Wilk test)
indicate the most preferred sample. and were subsequently analyzed using nonparametric tests (Kruskal-
- Once the three most appropriate beverage were singled out, they Wallis) to identify differences among samples.
were submitted to a second sensory analysis with the objective of To evaluate the influence of a selection of physicochemical param­
selecting the product with the best acceptance. The non-trained eters on the sensorial evaluation, the data were analyzed by multiple
panel consisted of thirty tasters randomly recruited at the univer­ regression analysis.
sity campus (18 women and 12 men, between 21 and 54 years old). All statistical analyses were performed using the program Statistica 8
Hedonic test and rank-order test were the two procedures used. In for Windows (StatSoft, 2007).
the hedonic test, judges were asked to evaluate how much they liked
the beverages using a 7-points hedonic scale (1 = ’Strongly disliked’; 3. Results and discussion
2 = ’Moderately disliked’; 3 = ’Slightly disliked’; 4 = ’Indifferent’; 5
= ’Slightly liked’; 6 = ’Moderately liked’ and 7 = ’Strongly liked’) to 3.1. Physicochemical properties
determine if there were differences between products in judge’s
evaluation (Drake, 2007; Granato et al., 2010). Additionally, a Table 2 shows the results of the physicochemical analyses.
rank-order test was carried out to evaluate the overall preference The metabolism rate of fermentable sugars increased with the
considering attributes such as aroma, texture and colour. fermentation time, decreasing the content of soluble solids (◦ Brix). The
highest decrease in ◦ Brix was observed in the beverage fermented with
1% w/v kefir grain during 48 h (from 10.91 to 3.62 ◦ Brix) and the lowest
2.4. Statistical analysis in the samples fermented during 12 h (from 10.91 to 10.27 ◦ Brix)
(Table 2). Corona et al. (2016) reported a sugar reduction (in ◦ Brix) in
All experiments were done in triplicate and the data are presented as kefir-fermented juices varying from 6.22 in melon and 4.77 in carrot to
the mean and standard deviation of three independent experiments. The 3.48 in strawberry juices. Similar results were reported by Randazzo
statistical analysis of data was performed by one-way analysis of

3
 J.L. Paredes et al.
Table 2
Physico-chemical analysis of samples (mean ± standard deviation) fermented with different kefir inoculum concentrations (1%, 2%, 3% and 4% w/v), during different fermentation times (12, 24 and 48 h).
Fermentation 0H 12H 24H 48H
Time

%Kefir C 1% 2% 3% 4% 1% 2% 3% 4% 1% 2% 3% 4%
Inoculum

SSC (◦ Brix) 10.91 ± 0.21 10.27 ± 0.13 9.79 ± 0.11 10.10 ± 0.12 9.83 ± 0.10 9.93 ± 0.04 9.87 ± 0.04 8.69 ± 0.04 9.19 ± 0.36 3.62 ± 0.04 3.81 ± 0.19 4.13 ± 0.18 4.06 ± 0.16
1a 2b 2c 2b 2c 3b 2b 3c 3c 4b 3bc 4c 4c
CO2 (g/100 mL) n.d 0.45 ± 0.07 0.52 ± 0.02 0.46 ± 0.03 1.16 ± 0.03 1.23 ± 0.02 0.70 ± 0.01 0.68 ± 0.02 1.35 ± 0.02 4.09 ± 0.01 4.28 ± 0.04 4.29 ± 0.03 4.31 ± 0.03
1a 2bc 2b 2c 2d 3b 3c 3c 3d 4b 4c 4c 4c
pH 3.80 ± 0.04 3.65 ± 0.01 3.60 ± 0.01 3.62 ± 0.02 3.53 ± 0.03 3.70 ± 0.03 3.67 ± 0.03 3.65 ± 0.01 3.64 ± 0.01 3.48 ± 0.02 3.41 ± 0.01 3.40 ± 0.03 3.39 ± 0.02
1a 2b 2c 2bc 2d 2b 3bc 2bc 3c 3b 4c 3c 4c
TTA (g/L Malic 4.00 ± 0.08 4.18 ± 0.04 4.40 ± 0.10 5.19 ± 0.05 5.27 ± 0.04 4.78 ± 0.08 5.36 ± 0.13 5.64 ± 0.07 6.28 ± 0.27 5.49 ± 0.07 6.12 ± 0.14 6.95 ± 0.29 6.86 ± 0.10
Acid)
1a 2b 2c 2d 2d 3b 3c 3d 3e 4b 4c 4d 4d
Viscosity (CP) 2.78 ± 0.30 3.47 ± 0.13 2.97 ± 0.17 2.92 ± 0.02 2.80 ± 0.02 3.40 ± 0.03 2.55 ± 0.01 2.64 ± 0.01 2.78 ± 0.01 2.66 ± 0.04 2.52 ± 0.01 2.53 ± 0.11 2.52 ± 0.04
1a 2b 1acd 2c 1d 2b 2c 3d 1e 3b 2c 3bc 2c
Density (g/mL) 1.0287 ± 1.0283 ± 1.0280 ± 1.0247 ± 1.0267 ± 1.0230 ± 1.0263 ± 1.0257 ± 1.0267 ± 1.0137 ± 1.0087 ± 1.0140 ± 1.0130 ±
0.0025 0.0006 0.0017 0.0021 0.0015 0.0035 0.0012 0.0012 0.0021 0.0006 0.0067 0.0020 0.0069
1a 12a 12ab 2b 1ab 2b 2b 2b 1ab 3b 3b 3b 2b
TP (mg/L) 1051.97 ± 1046.69 ± 1081.83 ± 1276.22 ± 1228.22 ± 1155.28 ± 1381.03 ± 1065.17 ± 1151.44 ± 1046.25 ± 1167.89 ± 1061.50 ± 853.39 ±
14.47 42.20 44.29 34.11 33.80 24.88 39.30 31.16 36.95 26.29 27.24 13.39 30.20
1a 1a 1a 2b 2b 2b 2c 1a 2b 1a 3b 1a 3c
ABTS (umoles/L) 684.33 ± 443.24 ± 370.82 ± 344.42 ± 421.61 ± 341.55 ± 420.37 ± 419.35 ± 391.39 ± 427.87 ± 490.69 ± 444.21 ± 447.19 ±
7.51 7.61 59.93 5.50 6.09 7.79 9.95 6.03 10.42 7.53 5.06 5.52 4.71
1a 2b 2bcd 2c 2d 3b 2c 3c 3d 2b 3c 4d 4d
Lactic Acid (mg/ n.d 289.61 ± 631.00 ± 899.49 ± 1479.92 ± 515.45 ± 1099.94 ± 1609.50 ± 2042.28 ± 559.11 ± 1094.25 ± 2739.09 ± 3250.47 ±
4

L) 15.68 26.67 20.04 22.85 20.72 23.95 71.12 9.75 60.65 71.62 70.75 76.87
1a 2b 2c 2d 2e 3b 3c 3d 3e 3b 3c 4d 4e
Acetic Acid (mg/ n.d n.d n.d 62.07 ± 6.65 85.40 ± n.d 37.01 ± 3.79 96.39 ± 8.02 98.32 ± n.d 60.73 ± 4.52 248.49 ± 266.81 ±
L) 20.24 15.66 25.14 16.79
1a 1a 1a 2d 2d 1a 2b 3c 2c 1a 3b 4c 3c
Citric Acid (mg/ 899.86 ± 682.86 ± 614.80 ± 542.84 ± 498.22 ± 607.55 ± 543.96 ± 499.33 ± 496.54 ± 594.17 ± 601.42 ± 567.95 ± 532.80 ±
L) 68.53 12.34 39.26 19.61 18.64 23.45 10.76 22.84 27.85 21.78 36.45 49.34 27.21
1a 2b 2c 2d 2e 3b 3c 2d 2cd 3b 23bc 2bc 2c
Succinic Acid 234.43 ± 249.39 ± 352.99 ± 279.29 ± 201.32 ± 364.73 ± 273.95 ± 287.84 ± 267.54 ± 380.75 ± 264.34 ± 369.01 ± 379.69 ±
(mg/L) 33.66 11.25 16.44 14.45 22.73 21.33 11.55 15.14 30.90 24.26 12.82 31.61 12.82
1a 1b 2c 1a 2d 2b 1a 1a 1a 2b 1a 2b 3b
Malic Acid (mg/ 832.37 ± 874.41 ± 871.86 ± 1005.01 ± 1190.39 ± 1421.63 ± 1105.66 ± 1449.03 ± 1210.14 ± 1552.87 ± 2244.70 ± 1728.69 ± 1759.90 ±
L) 55.27 29.00 17.76 90.37 39.91 66.89 47.48 33.99 37.56 63.45 64.14 17.24 26.76
1a 2b 2b 2b 2c 3b 3c 3b 2d 3b 4c 4d 3d
L* 29.52 ± 0.07 28.12 ± 0.01 28.36 ± 0.04 29.27 ± 0.07 28.56 ± 0.06 29.15 ± 0.09 30.52 ± 0.05 30.16 ± 0.46 30.36 ± 0.05 31.77 ± 0.05 31.27 ± 0.40 31.68 ± 0.10 30.92 ± 0.03
1a 2b 2c 2d 2e 3b 3c 1acd 3d 4b 4bc 3b 4c
C*ab 17.64 ± 0.06 13.51 ± 0.02 14.37 ± 0.10 17.04 ± 0.38 14.85 ± 0.05 16.72 ± 0.27 19.42 ± 0.12 19.03 ± 0.17 19.15 ± 0.60 22.53 ± 0.05 22.63 ± 0.53 22.78 ± 0.13 20.62 ± 0.01
1a 2b 2c 2d 2e 3b 3c 3d 3cd 4b 4bc 4c 4d
hab 14.49 ± 0.07 10.88 ± 0.16 10.92 ± 0.21 13.64 ± 0.24 11.02 ± 0.25 13.30 ± 0.07 14.87 ± 0.12 13.40 ± 0.03 13.33 ± 0.23 13.28 ± 0.03 13.07 ± 0.01 12.56 ± 0.01 12.41 ± 0.02
1a 2b 2b 2c 2b 3b 3c 2b 3b 3b 4c 3d 4e
DE*ab 0 4.52 ± 0.10 3.66 ± 0.03 0.75 ± 0.28 3.16 ± 0.08 1.12 ± 0.36 1.99 ± 0.18 1.56 ± 0.26 1.73 ± 0.14 5.35 ± 0.14 5.27 ± 0.43 5.56 ± 0.16 3.30 ± 0.11

Mean values of three measurements for each replicate.

LWT 154 (2022) 112728

C: control beverage; SSC: soluble solid content; CO2: carbon dioxide; TTA: total titratable acidity; TP: total phenol (gallic acid equivalent mg/L); ABTS: Trolox equivalent antioxidant activity (TEAC: Trolox-equivalent
antioxidant capacity, μmoles de Trolox/L); L*: lightness; hab: hue; C*ab: chroma; n.d.: not detectable.
Different letters in the same file indicate significant differences between samples with different concentration inoculum of kefir for each time fermentation time (p < 0.05).
Different numbers in the same file indicate significant differences between samples with different fermentation times for each concentration of kefir (p < 0.05).
J.L. Paredes et al. LWT 154 (2022) 112728

et al. (2016) for grape pomegranate and quince juices (6.46, 6.36 and Succinic acid showed an oscillating tendency and, except for 2% w/
5.8 ◦ Brix reductions). The high sucrose content of the fruit-vegetable v kefir, succinic acid increased its concentration with the fermentation
juice probably stimulates the growth of Saccharomyces species, which time (from 12 to 48 h). The values of succinic acid for the sample with
can hydrolyze sucrose into glucose and fructose by the enzyme inver­ 1% w/v ranged between 249.4 and 380.8 mg/L, being the last one the
tase, making this carbon source available to lactic acid bacteria (Fiorda highest concentration at 48 h. This is in accordance with results reported
et al., 2017). by Texeira et al. (2010).
Accordingly, Carbon dioxide (CO2) production was also related to the For malic acid, it was observed an increase in concentration with
fermentation times and significant differences (p ≤ 0.05) among them increasing fermentation times. However, this tendency was not found
were detected. The highest CO2 production (4.31 g CO2/100 mL) was for increasing concentrations of kefir. Therefore, after 48 h of fermen­
found in the beverage fermented with 4% w/v of kefir grains for 48 h. tation, the highest malic acid concentration was obtained for the sample
This behaviour is related to the decrease of soluble solids content during fermented with kefir at 2% w/v (2244.7 mg/L). Sabokbar et al. (2015),
the fermentation time. Corona et al. (2016) and Randazzo et al. (2016), reported a reduction in malic acid concentration in a mixture of apple
on similar fermentation conditions in melon and pomegranate juices, juice and whey fermented with kefir.
reported lower amounts of CO2 production (3.39 g CO2/100 mL and However, the increase in malic acid observed could be due to the
3.21 g CO2/100 mL respectively). CO2 is the major fermentation product presence of Saccharomyces cerevisiae in the microbiota of the selected
of yeasts that contributes to the desirable exotic notes and yeast flavor kefir since it is one of the microorganisms capable of producing malic
(Güzel-Seydim, Seydim, Greene, & Bodine, 2000). acid (Chi, Wang, Wang, Khan, & Chi, 2016).
Significant differences (p < 0.05) in pH and Titulable Total Acidity The results shown in Table 2 indicate that the viscosity increased only
(TTA) among samples with different fermentation times were detected. in the first 12 h of fermentation. At that point, the viscosity tends to
The highest increase in TTA and the lower pH value was observed for the decrease as the fermentation time increases. Consequently, the highest
sample fermented 48 h with 3% w/v of kefir grains which had 6.95 g of viscosity value was observed in the sample at 1% w/v of kefir grains
malic acid/L and pH 3,4, this is in accordance with previously published during the first 12 h of fermentation with a value of 3.47 CP. On the
results in kefir fermented fig juice (Corona et al., 2016). The production other hand, the variation of the content of kefir grain also had a sig­
of organic acids in fermented foods reduces the pH value and increases nificant effect on the viscosity.
total titratable acidity (Puerari, Magalhaytild, Guedes, & Schwan, The increase in viscosity in the first 12 h of fermentation is related to
2015). According to Anton et al. (2016), low pH values prevent the lactobacilli which reform the internal structure of the beverages
growth of most waste and pathogenic organisms, also create a suitable improving the consistency of kefir and causing greater resistance of the
environment for the growth of yeasts and probiotic lactic acid bacteria. inner layer of the drink and consequently a higher viscosity (Irigoyen,
The organic acids analyzed were lactic, acetic, citric, succinic, and Arana, Castiella, Torre, & Ibáñez, 2005). However, Degeest, Mozzi, and
malic acid (Table 2). The concentration of lactic acid increased signif­ De Vuyst (2002) stated that glycohydrolases may hydrolyze exopoly­
icantly (p < 0.05) with the fermentation time and the content of kefir saccharide (EPS) material in their monomers explaining the decrease in
grains. Therefore, at a fermentation time of 48 h and 4% w/v of kefir viscosity detected afterwards. Glycohydrolases can decrease the vis­
grains, the lactic acid reached the highest concentration with a value of cosity of the polymers produced by Lactobacillus rhamnosus, as well as
3250.5 mg/L. The production of lactic acid during fermentation is release some reducing sugars (Pham, Dupont, Roy, Lapointe, & Cerning,
attributed to the metabolism of lactic acid bacteria. According to Puer­ 2000).
ari, Magalhães-Guedes, and Schwan (2015), lactic acid is the result of As shown in Table 2 the density of the juices decreases as the
homofermentative metabolism, and it is of great importance due to the fermentation time increases, this decrease was significant (p < 0.05) in
inhibitory effect on pathogenic microorganisms (Texeira, Pereira, some cases, except for the samples between 12 and 24 h of fermentation.
Ribeiro, & Freitas, 2010). These results may be related to the metabolization sugars which are
The first traces of acetic acid were detected after 12 h of fermen­ fermented into lactic acid, acetic acid, alcohol and carbon dioxide, and
tation in samples with 3% kefir w/v (62.1 mg/L). For concentrations of other compounds which may cause a change in the density of the
kefir above 2% w/v, the production of acetic acid was directly propor­ beverage.
tional to the fermentation time. Therefore, the maximum concentration Total phenols (TP) content of the samples changed significantly
of acetic acid was detected in kefir at 4% w/v, after 48 h of fermentation during the different times of fermentation (Table 2). The sample with
(266.8 mg/L.) Bellow 1% w/v of kefir, acetic acid production was not 4% w/v of kéfir grains and 48 h of fermentation was the one with the
detected in accordance with Bensmira and Jiang (2011) and Texeira highest reduction in TP concentration (decrease: 19%). The sample at
et al. (2010) who reported that the average concentration of acetic acid 2% w/v of kefir grains in 24 h of fermentation reached the highest
was practically zero during the first 18–24 h of fermentation of peanut concentration of TP (increase: 31%). Randazzo et al. (2016) reported a
milk and whey in kefir. When the lactic and acetic acid bacteria present decrease in TP in kiwi, pomegranate, strawberry, apple, grape and
in the kefir microflora use the heterofermentative route, an increase in quince juices in similar fermentation conditions as in this research (4%
the amount of acetic acid and, in smaller proportion, of succinic, formic w/v kéfir, 48 h of fermentation). According to McCue and Shetty (2005)
acid and carbon dioxide, among others are detected (Texeira et al., the decrease in TP content could be the result of the degradation of
2010). In this case, likely the preferred metabolic route of fermentation phenolic structures as possible mechanisms of antimicrobial detoxifi­
of the kefir microflora in the commercial product was cation of yeasts and bacteria.
homofermentative. The fermentation time had a significant effect on the antioxidant
The citric acid decreased as the concentration of kefir grains activity (AA) values of the samples (p < 0.05). The results reported in
increased and the effect of the fermentation time followed this trend Table 2 show a decrease between 28% (490.69 μmol TE/L) and 37%
only in 1% w/v kefir. Samples with 3 and 4% w/v kefir during the first (427.87 μmol TE/L) after 48 h of fermentation on the AA compared to
24 h of fermentation decreased the concentration of citric acid to a value the control beverage (684.33 μmol TE/L). Randazzo et al. (2016) re­
of 499.3 and 496.5 mg/L respectively, representing the lowest registered ported similar results, with a decrease of AA in pomegranate, grape,
values; followed by the sample with 4% w/v kefir during 48 h of apple, kiwi and fig juice, only the quince juice showed a slight increase
fermentation (532.8 m/L). Similarly, Sabokbar et al. (2015) reported in AA. According to McCue and Shetty (2005) there is an inverse relation
that the level of citric acid decreased during the fermentation of apple between total phenolic content and the antioxidant activity of the kefir
juice and whey with kefir, and Bensmira and Jiang (2011) showed that fermented beverage, as observed in the present investigation.
the level of citric acid decreased due to some lactic acid bacteria which
prefer citric acid as a substrate to produce acetoin and diacetyl.

5
J.L. Paredes et al. LWT 154 (2022) 112728

3.2. Colour analysis time increases. That is, the fermentation time provides lighter and more
reddish beverages.
The location of the samples within the (a*b*) plane is shown in The variation of kefir grain concentrations only had a significant
Fig. 2. All the samples are clustered in the red area of the diagram, with effect (p < 0.05) on the values of L* and C*ab after the first 12 h of
values of a* and b* around 14–22 and 2–4 CIELAB units, respectively. fermentation, and for hab values after 48 h of fermentation.
Colour parameters L*, C*ab, and hab were significantly affected by the Randazzo et al. (2016) state that the lightness reduction and the
fermentation time (Table 2). The results show a significant decrease of reddish increase could be explained by the browning processes that
values (p < 0.05) in colourimetrics parameters L*, C*ab and hab during occur during the fermentation of these beverages. This phenomenon is
the first 12 h of fermentation with respect to the control beverage due to the activation of oxidase enzymes, such as polyphenol oxidase,
(0.8–4.7%, 3.5–23.4% and 5.8–24.9%, respectively). However, after 48 when the environments are not completely anaerobic (Corona et al.,
h of fermentation, the samples significantly increased their L* and C*ab 2016). The above confirms the variation in L*, C*ab and hab observed in
values with respect to the control beverage (4.7–7.6% and 16.8–29.1% the samples due to the fermentation time. The colour differences be­
respectively). The hue values (hab) of the samples tend to stabilize as tween the control (raw juice) and the fermented samples were deter­
mined. It was found that the beverages fermented during 48 h presented
the highest colour difference values (ΔE*ab = 3.30–5.56 CIELAB units)
which could be perceived by the human eye, according to Martínez,
Melgosa, Pérez, Hita, and Negueruela (2001), which indicate that ΔE*ab
values above 2.7 CIELAB can be clearly detected by a non-trained
human eye.

3.3. Sensorial analysis

Results from the two sensorial analyses are described in this section:
the aim of the first one was to choose the three best beverages from 12
samples consisting of beverages with three levels of fermentation time
and four levels of kefir grain concentration, and the second one was to
select the best beverage amongst the three samples chosen in the pre­
vious sensorial analysis.

3.3.1. Sensory analysis to assess the effect of fermentation time and kefir
concentration on sensorial properties of the beverages
The sample rank-sum was calculated, and the friedman test was used

Table 3
Sensory analysis to assess the effect of fermentation time on sensorial properties
of beverages. Summary of the judges’ answers in the rank-order tests and the
Friedman’s test results.
Fixed Attribute Rank sums F Fcritical Fcritical
variable (nº Samplesa (Friedman) (α = (α =
judges/ [12h]/ 0.05) 0.01)
session) [24h]/[48h]

1% w/v Kefir Sweet [37]a/[35]a/ 14.53** 6.40 8.93

(15) [18]b
Acidity [25]/[29]/ 4.13
[36]
Alcohol [26]/[28,5]/ 3.23
[35,5]

2% w/v kefir Sweet [29]a/[43]b/ 20.93**

(15) [18]c
Acidity [29]ab/ 7.60*
[23]a/[38]b
Alcohol [32]ab/ 6.93*
[22]a/[36]b

3% w/v kefir Sweet [38]a/[35]a/ 17.20**

(15) [17]b
Acidity [28.5]/ 1.63
[27.5]/[34]
Alcohol [29]/[25]/ 4.13
[36]

4% w/v kefir Sweet [35.5]a/ 17.03**

(15) [37.5]a/
[17]b
Acidity [28]/[28]/ 1.60
[34]
Alcohol [29.5]/ 3.03
[25.5]/[35]
a
Different superscripts in the same row indicate significant differences, *p <
Fig. 2. Location of the samples within the (a*b*) plane. 0.05 and **p < 0.01.

6
J.L. Paredes et al. LWT 154 (2022) 112728

to verify significant differences among the samples. Tables 3–5 present Table 5
the Friedman test statistic for each attribute (perception of sweetness, Sensory analysis to assess the effect of kefir concentration on sensorial properties
acidity, and alcohol). When significant differences among samples were of beverages. Summary of the judges’ answers in the rank-order tests and the
found, the pairs of differing samples were computed according to the Friedman’s test results.
minimal significant differences of Fisher (Tables 4–6). Fixed variable Attribute Rank sums F Fcritical Fcritical
Tables 3 and 4 shows the results of evaluating the effects of (nº judges/ Samples* (Friedman) (α = (α =
session) [1%]/ 0.05) 0.01)
fermentation time on the sensory properties. The Friedman’s test
[2%]/
applied to the ranking test revealed that, regardless of the concentration [3%]/[4%]
of kefir inoculum, the samples with different fermentation times (12, 24
12h Sweet [50]a/ 8.44* 7.81 11.34
and 48 h) were significantly different in terms of sweetness perception fermentation [34]b/
(F1% = 14.5, F2% = 20.9, F3% = 17.2, F4% = 17.0; Fcritical = 8.9, p < time (15) [34]b/[32]b
0.01). The judges have a significantly greater perception of sweetness in Acidity [35]/ 6.26
samples of 12 and 24 h of fermentation time than in samples of 48 h. The [35.5]/
[31.5]/[48]
sample corresponding to a fermentation time of 24 h and a concentra­
Alcohol [33]/ 2.34
tion of kefir of 2% w/v presented significantly the highest score in terms [43.5]/
of sweetness perception (judges score = 2.9). The results of the rank- [37.5]/[36]
order test related to the perception of acidity and alcohol in the prod­ 24h Sweet [38]/[47]/ 5.80
ucts allowed to determine that the judges perceived greater acidity and fermentation [34]/[31]
alcohol in the samples with 48 h of fermentation. Significant differences time (15) Acidity [26]a/ 19.00**
were found only between samples containing 2% w/v kefir and different [28]a/
[44]b/[52]b
fermentation times (acidity: F = 7.6 and alcohol: F = 6.9; Fcritical = 6.4, p
Alcohol [31.5]/ 5.84
< 0.05). The sample with fermentation time of 48 h and a concentration [32.5]/
of kefir of 2% w/v presented significantly the highest score in the acidity [46.5]/
and alcohol perception (judges score = 2.5 and 2.4, respectively). This [39.5]
result is in accordance with the production of organic acids as it can be 48h Sweet [52.5]a/ 8.80*
observed in Table 2, significant differences were found (p < 0.05) for fermentation [29.5]b/
samples inoculated at 2% w/v of kefir and different fermentation times. time (15) [37.5]b/
[28.5]b
On the other hand, the sample with the significantly lowest perception
Acidity [28]/ 5.02
of alcohol by the tasters was the sample at 2% w/v kefir fermented for [42.5]/
24 h (judges score = 1.5). [39.5]/[40]
Tables 5 and 6 shows the results of the sensory analysis to assess the Alcohol [26.5]/ 7.44
effect of kefir concentration on the sensory properties. The Friedman’s [44.5]/
[37.5]/
test applied to the ranking test revealed that, in the cases with 12 and 48 [41.5]
h of fermentation times like fixed variable, the samples with different
(1)
kefir concentrations (1, 2, 3 and 4% w/v) were significantly different in Different superscripts in the same row indicate significant differences, *p <
0.05 and **p < 0.01.
terms of sweetness perception (F12h = 8.4, F48h = 8.8; Fcritical = 7.8, p <
0.05). The judges have significantly greater perception of sweetness in
samples with 1% w/v of kefir than in samples with 2, 3 and 4% w/v of 2.1, 2.1 and 1.9 respectively). The results of the rank-order test related
kefir. The sample corresponding to kefir of 1% w/v and a fermentation to the perception of the acidity allow to determine that the judges
time of 48 h was scored significantly as the highest in terms of sweetness perceived greater acidity in the samples with 4% w/v kefir. Significant
perception (judges score = 3.5); and the samples with the lowest differences were found only between samples fermented for 24 h and
perception of sweetness by the tasters were the ones corresponding to containing different kefir concentrations (F = 19; Fcritical = 11.34, p <
4% w/v kefir with 12, 24 and 48 h of fermentation time (judges score = 0.01). The sample with 4% w/v of kefir and fermented for 24 h was
scored significantly as the highest in the acidity perception (judges
score = 3.5). In the case of the alcohol perception, according to Fried­
Table 4 man’s test results, samples were not statistically different.
Pair comparison test using minimal significant differences of Fisher. Difference In each session judges were asked to answer “What sample do you
between the rank sums in the formulations studied. This value compares the prefer?”. One point was assigned to the favorite sample and 0 points to
perceived differences in attributes between two fermentation times.
the others. All the points were summed up and divided by the number of
Fixed variable Attribute 12–24 12–48 24–48 LSD0.05 LSD0.01 judges to obtain the final score of each sample. The results of the pref­
(nº judges/ h h h
erence test (Fig. 3) indicate that the tasters had a greater preference for
session)
the samples that have been fermented for 24 h at 1%, 2% and 3% w/v
1% w/v Kefir Sweet 2 19** 17** 10.74 14.11 kefir. Therefore, these results show that the judges have preference for a
(15) Acidity 4 11* 7
product with an acidity between 4.78 and 5.64 g/L of malic acid, pH
Alcohol 2.5 9.5 7
between 3.65 and 3.7, refractive index between 8.69 and 9.93 ◦ Brix. In
2% w/v kefir Sweet 14* 11* 25**
addition, the judges preferred a product with low alcohol perception.
(15) Acidity 6 9 15**
Alcohol 10 4 14*
3.3.2. Sensory analysis to select the product with the best acceptance
3% w/v kefir Sweet 3 21** 18**
(15) Acidity 1 5.5 6.5
Only the beverages preferred by the judges in the previous assay,
Alcohol 4 7 11* were included: samples with 1%, 2% and 3% w/v kefir and 24 h of
fermentation, and the control beverage.
4% w/v kefir Sweet 2 18.5** 20.5**
(15) Acidity 0 6 6 Table 7 presents the Friedman test statistic for each attribute (aroma,
Alcohol 4 5.5 9.5 texture and colour). When significant differences among samples were
found, the pairs of differing samples were computed according to the
LSD: Least significant difference.
Significant difference between two fermentation times, *p < 0.05 and **p <
minimal significant differences of Fisher. The Friedman’s test applied to
0.01. the ranking test revealed that the samples were significantly different in

7
J.L. Paredes et al. LWT 154 (2022) 112728

Table 6
Pair comparison test using minimal significant differences of Fisher. Difference between the rank sums in the formulations studied. This value compares the perceived
differences in attributes between two kefir concentrations.
Fixed variable (nº judges/session) Attribute 1–2% 1–3% 1–4% 2–3% 2–4% 3–4% LSD0.05 LSD0.01

12h fermentation time (15) Sweet 16* 16* 18* 0 2 2 13.86 18.22
Acidity 0.5 3.5 13 4 12.5 16.5*
Alcohol 10.5 4.5 2 6 7.5 1.5
24h fermentation time (15) Sweet 9 4 7 13 16* 3
Acidity 2 18* 26** 16* 24** 8
Alcohol 1 15* 8 14* 7 7
48h fermentation time (15) Sweet 23** 15* 24** 8 1 9
Acidity 14.5* 11.5 12 3 2.5 0.5
Alcohol 18* 11 15* 7 3 4

LSD: Least significant difference.

Significant difference between the two kefir concentrations, *p < 0.05 and **p < 0.01.

Fig. 3. Summary of the judges’ answers in the preference test.

score was significantly higher than the one of the other analized bev­
Table 7
erages, including the control sample (the original juice). Besides, this
Sensory analysis to assess the consumer’s preference on beverages. Summary of
sample stands out from the other samples analized on attributes such as
the judges’ answers in the rank-order tests and the Friedman’s test results.
low acidity, high perception of sweetness, low alcohol perception and
nº Attribute Rank sums F Fcritical Fcritical
better preference about texture. Consequently, this product has a higher
judges/ Samplesa (Friedman) (α = (α =
session [1K24H]/ 0.05) 0.01)
probability of having a better acceptance by consumers.
[2K24H]/
[3K24H]/[C]
3.4. Explorative multivariate analysis of physicochemical and sensory
30 Aroma [92.5]a/ 9.66* 7.81 11.34
data
[74.5]b/
[70.5]b/[62.5]b
Texture [77]ab/[91]b/ 8.69* To assess the influence of the most relevant physicochemical pa­
[68.5]a/[63.5]a rameters on the sensory evaluation, a multiple regression analysis was
Color [81]/[71.5]/ 5.23 applied. The sensory attributes and general preference were considered
[63.5]/[84]
as dependent variables, and the physicochemical parameters, as inde­
a
Different superscripts in the same row indicate significant differences, *p < pendent ones. The results are summarized in Table 8. The variables
0.05. Pair comparison test using minimal significant differences of Fisher. selected by the forward stepwise analyses, explained more than 87% of
the sensorial analysis variations of the beverages analyzed, and the F of
terms of aroma and texture preference (F = 9.66 and F = 8.69 respec­ each regression was statistically significant (p < 0.05). High correlations
tively, Fcritical = 7.81, p < 0.05). The judges had significantly greater (R2 > 0,96) among the selected physicochemical parameters and sen­
preference for the aroma and texture of the samples corresponding to a sory attributes were obtained for sweetness, aroma, texture, colour and
fermentation time of 24 h and kefir concentration of 1% and 2% w/v preference.
(judges score for aroma = 3.1 and texture = 3.0). Regarding the colour ◦
Brix, showed a significant (p < 0.05) and positive correlation with
attribute, there were not significant differences (p < 0.05) among the sweetness (b = 1211), aroma (b = 0,244) and colour (b = 1888), and
judges’ preferences. was also negatively related to alcohol perception. CO2 was significant (p
Fig. 4 shows the results of the preference hedonic test. The Kruskal- < 0.05) and positively correlated with aroma (b = 1046), texture (b =
Wallis test was applied to identify differences among samples. The 1392), colour (b = 0,335) and preference (b = 1237). pH and TTA were
product at 2% w/v of kefir [2K24H] got the highest score (5.37). This related to acidity, alcohol perception, texture, and preference while
viscosity and density were selected variables to predict alcohol, aroma,

8
J.L. Paredes et al. LWT 154 (2022) 112728

Fig. 4. Mean values preference scores given by non-trained panel (Bar with different letters indicate significant differences between samples, p < 0.05).

Fundings
Table 8
Summary of the regression analyses carried out considering sensory attributes as
Jorge Luis Paredes enjoyed a Foreign Master’s Scholarship from
dependent variables and the physicochemical parameters as independent
variables.
Gobernación del Putumayo-Colciencias [754-Formación Capital
Humano].
Dependent R2 Independent variables selected by the forward stepwise
variable analysesa
CRediT authorship contribution statement
Sweet 0.96 ◦
Brix, C*ab, L*, hab
Acidity 0.88 ◦
Brix, L*, C*ab, CO2, TTA
Alcohol 0.87 pH, Viscosity, ◦ Brix, L*, TTA, CO2 Jorge Luís Paredes: Methodology, Formal analysis, Writing – orig­
Aroma 0.99 CO2, TTA, ◦ Brix, C*ab, Density inal draft. María Luisa Escudero-Gilete: Conceptualization, Method­
Texture 0.99 CO2, hab, C*ab, pH, L*, TTA, Viscosity ology, Data curation, Writing – review & editing, Project administration.
Colour 0.99 TTA, CO2, ◦ Brix, C*ab, Viscosity, L*, Density, hab, pH
Isabel María Vicario: Resources, Visualization, Writing – review &
Preference 0.99 TTA, ◦ Brix, CO2, hab, C*ab, pH, L*, Viscosity
editing, Project administration.
TTA: total titratable acidity.
a
The variables in italics present significant correlation coefficients (p < 0.05).
Declaration of competing interest
texture, colour and preference. It is interesting to note the relevance of
The authors declare that they have no known competing financial
colour parameters (hab, C*ab, L*) in the sensory attributes evaluated.
interests or personal relationships that could have appeared to influence
Chroma, hue and lightness of the beverages showed a significant and
the work reported in this paper.
positive correlation (b = 0,568; 0,865; 0,178) with the preference.
Preference was also affected by ◦ Brix, TTA and viscosity.
Acknowledgements
4. Conclusions
Servicios Generales de Investigación, U. Sevilla.
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