Journal Pre-proof

Powdered water kefir: effect of spray drying and lyophilization on

physical, physicochemical, and microbiological properties

Klinger Vinı́cius de Almeida , Vanessa Cortina Zanetti ,

Callebe Camelo-Silva , Luan Amaral Alexandre ,
Alice Cristina da Silva , Silvani Verruck ,
LucianoLuciano José Quintão Teixeira

PII: S2772-753X(24)00155-2
DOI: https://doi.org/10.1016/j.focha.2024.100759
Reference: FOCHA 100759

To appear in: Food Chemistry Advances

Received date: 20 February 2024

Revised date: 6 June 2024
Accepted date: 13 June 2024

Please cite this article as: Klinger Vinı́cius de Almeida , Vanessa Cortina Zanetti ,
Callebe Camelo-Silva , Luan Amaral Alexandre , Alice Cristina da Silva , Silvani Verruck ,
LucianoLuciano José Quintão Teixeira , Powdered water kefir: effect of spray drying and lyophilization
on physical, physicochemical, and microbiological properties, Food Chemistry Advances (2024), doi:
https://doi.org/10.1016/j.focha.2024.100759

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

© 2024 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/)
 1

Highlights

 The ideal fermentation condition was using 5% grains, 10% sugar and 25°C.
 Freeze-drying preserved microbial viability better than spray-drying.
 Spray-dried kefir powders showed better solubility compared to lyophilized
powders.
 Zymomonas mobilis dominated kefir microbiota, constituting 94.31% of grains
 2

Powdered water kefir: effect of spray drying and lyophilization on physical,

physicochemical, and microbiological properties

Klinger Vinícius de Almeidaa, Vanessa Cortina Zanettib, Callebe Camelo-Silvac, Luan

Amaral Alexandreb, Alice Cristina da Silvab, Silvani Verruckb, Luciano José Quintão

Teixeiraa*

a
Departamento de Engenharia de Alimentos, Centro de Ciências Agrárias e

Engenharias, Universidade Federal do Espírito Santo, Alegre, ES, Brazil

b
Departamento de Ciência e Tecnologia de Alimentos, Centro de Ciências Agrárias,

Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil

c
Departamento de Engenharia Química e de Alimentos, Centro Tecnológico,

Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil

*Corresponding author:

Prof. Dr. Silvani Verruck

Departamento de Ciência e Tecnologia de Alimentos, Centro de Ciências Agrárias,

Universidade Federal de Santa Catarina, Avenida Ademar Gonzaga, 1346, 88034-001,

Itacorubi, Florianópolis, SC, Brazil

E-mail address: silvani.verruck@ufsc.br

Phone number: +55 49 998089107

 3

Abstract

This study addresses the challenge of optimizing powdered water kefir's fermentation

and preservation processes to enhance its physical-chemical, microbiological, and

technological characteristics. The main objective was to determine the best fermentation

conditions and evaluate the efficacy of different drying methods. The optimal

fermentation conditions were 5% kefir grains, 10% brown sugar, and an incubation

temperature of 25°C. Remarkably, the microbiological analysis revealed high

abundances of Zymomonas mobilis (grains: 94.31% and beverage: 91.68%),

Sporolactobacillus spathodeae (grains: 3.00% and beverage: 5.42%), and

Liquorilactobacillus satsumensis (grains: 1.47% and beverage: 0.62%) among bacteria,

and Lachancea fermentati (grains: 95.54% and beverage: 67.53%), Wickerhamomyces

anomalus (grains: 3.00% and beverage: 26.77%) among fungi. The study innovatively

demonstrates that lyophilization preserves the viability of these microorganisms,

making it a promising method for producing stable, probiotic-rich powdered kefir.

Although spray drying resulted in a logarithmic reduction of 3 logs CFU/g, it

maintained sufficient microorganism counts, proving its viability as an alternative

drying method. These methods retain the ideal physical-chemical properties and expand

the accessibility and practical applications of water kefir. This research underscores the

potential for powdered water kefir to deliver health benefits conveniently and

versatilely, paving the way for broader industrial and academic applications.

Keywords: metataxonomics, spray dryer, lyophilization, kinetics, maltodextrin, inulin.

Abbreviations
 4

MRS - De Man, Rogosa, and Sharpe Agar; DRBC - Dichloran Rose Bengal

Chloramphenicol Agar; PCA - Plate Count Agar; CFU - Colony Forming Units; FTIR

- Fourier Transform Infrared Spectroscopy; DSC - Differential Scanning Calorimetry

ATR - Attenuated Total Reflection; ITS - Internal Transcribed Spacer; DNA -

Deoxyribonucleic Acid; PCR - Polymerase Chain Reaction; V3/V4 - Third and Fourth

Variable Regions (of the 16S rRNA gene); w/v - Weight/Volume (percentage).

1. Introduction

The development of new functional beverages is a rapidly growing sector in the

food industry, aiming to meet the demand for healthier products (de Souza, Monteiro, et

al., 2024). Water kefir, a non-dairy kefir, is produced by fermenting water inoculated

with kefir grains in a sucrose solution. Brown sugar is typically used as the main

substrate for water kefir fermentation (Guzel-Seydim et al., 2021). However, various

vegetables and fruits, such as soy, onion, ginger, carrot, apple, pineapple, grape, quince,

kiwi, pear, melon, strawberry, pomegranate, tomato, and coconut, can also be used as

substrates. (Corona et al., 2016; da Silva Fernandes et al., 2017; Fiorda et al., 2017;

Randazzo et al., 2016). These alternatives offer diversification, nutritional enrichment,

and sensory improvement of the product (Sanches et al., 2024).

Water kefir fermentation typically lasts two to three days at temperatures

between 20 and 25 °C, after which fruit juices or pulps may be added to enhance flavor

(Laureys and De Vuyst, 2014). The resulting fermented beverage is carbonated, cloudy,

slightly alcoholic, and sweet, making it particularly popular among vegan consumers.

The grain cultivation conditions and microbiological composition largely determine the

beverage's composition (Fiorda et al., 2017). The final fermentation products include
 5

ethanol, lactic acid, mannitol, acetic acid, glycerol, and other organic acids (Gulitz et al.,

2011).

The fermentation conditions, grain structure, and substrate differences result in

varying microbial compositions in water kefir. Typically, water kefir grains comprise

lactic and acetic acid bacteria from genera such as Lactobacillus, Acetobacter, and

Gluconobacter, along with yeasts from genera including Pichia, Saccharomyces,

Kazachstania, Candida, and Zygosaccharomyces, living in symbiosis (Guzel-Seydim et

al., 2021; Xu et al., 2019). Traditionally, kefir microflora has been studied using

culture-dependent techniques. However, the advent of metataxonomic methods has

provided a promising approach to understanding the microbial dynamics of this

complex biota (Ilıkkan and Bağdat, 2021; Yerlikaya et al., 2022).

Recent studies have explored various aspects of water kefir such as

physicochemical and sensorial parameters (Tireki, 2024), microbial viability and

nutritional content of water kefir grains under different storage conditions (Gökırmaklı

et al., 2024), metabolic spectrum characteristics and core metabolite interaction

networks during water kefir fermentation using non-targeted metabolomics (Ma et al.,

2024), functional quality of water kefir enriched with Haematococcus pluvialis biomass

and astaxanthin pigment (Yıldız et al., 2024), microbial composition of water kefir

grains and their application for detoxifying aflatoxin B1 (Ouyang et al., 2024), co-

fermentation of water kefir with Saccharomyces boulardii to develop a new probiotic

mead (de Souza, Bogáz, et al., 2024) and the characterization of a product based on

quinoa extract fermented with water kefir grains (Sanches et al., 2024).

Despite the numerous studies associated with water kefir, several challenges

persist, including the lengthy production process, difficulty maintaining grain viability,

and lack of product standardization (Gökırmaklı et al., 2024). To produce powdered

 6

kefir could be a viable alternative to address these issues, offering longer shelf life,

easier storage, and instant reconstitution, making it a convenient product (Teijeiro et al.,

2018). On the other hand, to our knowledge, no prior study has focused on developing

powdered water kefir, making this research pioneering in describing the influence of

drying on the physicochemical and microbial characteristics of water kefir.

Spray drying and lyophilization are the most widely used methods for producing

powdered foods (Atalar and Dervisoglu, 2015; Chaturvedi et al., 2021). However,

various intrinsic and extrinsic parameters influence the overall powder formulation

process. These include the microorganisms' resistance to heat and cold, oxygen

concentration, mechanical and osmotic forces exposure, and appropriate drying

parameters (Teijeiro et al., 2018). To preserve the viability of the microorganisms,

specific drying agents like starches and maltodextrins are added, along with

thermoprotective agents such as trehalose, glucose, inulin, and oligosaccharides

(Chaturvedi et al., 2021). Trehalose and glucose form a glassy matrix and maintain

hydrogen bonding, stabilizing proteins and membranes against dehydration and

temperature fluctuations (Olgenblum et al., 2020). Inulin, maltodextrin, and

oligosaccharides provide structural support and moisture retention, preserving cellular

integrity and enhancing thermal stability (Verruck et al., 2019).

This study aims to develop and analyze powdered water kefir's physical,

physicochemical, microbiological, and technological characteristics, using maltodextrin

and inulin as carrier agents during spray drying and lyophilization.

2. Materials and Methods

2.1. Materials
 7

The water kefir grains were acquired by donation from a local producer in the

Brazilian state of Santa Catarina. Water, brown sugar (Da Colônia, Santo Antônio da

Patrulha – RS, Brazil), inulin D.E. 10 (Beneo, Mannheim, Germany), and maltodextrin

D.E. 10 (Ingredion, Mogi Guaçu – SP, Brazil) were used in the production of water

kefir. De Man–Rogosa–Sharpe Agar – MRS (Merck, Germany), Dichloran Rose Bengal

Chloramphenicol Agar – DRBC (Merck, Germany), and Plate Count Agar – PCA

(Merck, Germany) were used for the microbiological assays. All reagents used were

analytical grade.

2.2. Maintenance of kefir grains

To maintain the viability of the kefir culture, the grains were inoculated and kept

in a solution of filtered water and brown sugar with soluble solids (SS) concentrations

of 10g/100g and a temperature of 25 ± 1 °C in a bacteriological culture oven

(SolidSteel, Brazil). The grains were kept active and viable for fermentation with

continuous change of the nutrient solution every 24 hours, as described by Alves et al.

(2021).

2.2. Fermentation kinetics of water kefir culture

In order to define the best fermentation parameters and subsequently submit the

fermented beverage to the drying processes, fermentation kinetics was constructed

following a completely randomized design in a 2x2x2 factorial scheme, where different

concentrations of grains and substrate (brown sugar) and different incubation

temperatures were evaluated (Table S1 – supplementary material). The fermentation

process lasted 48 hours.

 8

To choose the best fermentation condition, the following parameters were used:

the condition with the highest lactic bacteria count, the condition with the highest grain

mass growth, and finally (according to Equation 1), the condition that at the end of the

48 hours of fermentation reached a pH of 4.5, considered optimal for water kefir (Fiorda

et al., 2017).

𝑚𝑘𝑓−𝑚𝑓𝑖
𝑋(𝑔⁄100 𝑔) = ( ) × 100 (Eq. 1)
𝑚𝑘𝑖

Where:

mki = kefir grain starter mass

mkf = kefir grain mass recovered after fermentation

According to the data's behavior and non-linear regression, it was possible to

adjust mathematical models that describe the variation of the pH value as a function of

fermentation time (Equation 2).

𝑛
^ = 𝑦𝑒𝑞 + (𝑦0 − 𝑦𝑒𝑞 )𝑒 −𝑘𝑡
𝑦 (Eq. 2)

Where:

𝑦
^: estimated value for the pH response variable

𝑦0 , 𝑦𝑒𝑞 , 𝑘, and 𝑛: model parameters

2.3. Molecular microbial identification

After defining the best fermentation condition, the microbial diversity of the

kefir grains and the fermented beverage was studied based on sequenced libraries using

the MiSeq Sequencing System equipment (Illumina Inc., USA) and the V2 kit, with 300

cycles and single-end sequencing. For high-throughput sequencing of the V3/V4

regions of the 16S rRNA gene and the ITS1 and ITS2 regions of the ITS intergenic
 9

region, a 25 g/mL aliquot of the sample was homogenized with 225 mL of tryptone

saline. After this step, DNA extraction was performed using the magnetic bead

technique with a proprietary protocol developed by Neoprospecta Microbiome

Technologies, Brazil. Then, the PCR reaction was performed in triplicate using

Platinum Taq Polymerase (Invitrogen, USA) under the conditions: 95 °C for 5 minutes,

25 cycles of 95 °C for 45 seconds, 55 °C for 30 seconds, 72 °C for 45 seconds, and a

final extension at 72°C for 2 minutes. The sequences were analyzed using a pipeline and

library preparation following a proprietary protocol (Neoprospecta Microbiome

Technologies, Brazil). Amplification was performed with primers 341F

(CCTACGGGRSGCAGCAG) (Wang and Qian, 2009) and 806R

(GGACTACHVGGGTWTCTAAT) universal for the V3/V4 region of the 16S rRNA

gene (Caporaso et al., 2012). For fungi, amplification was generated with primers for

the ITS1 region, primer ITS1 (GAACCWGCGGARGGATCA) (Schmidt et al., 2013)

and primer ITS2 (GCTGCGTTCTTCATCGATGC) (White et al., 1990). The DNA

sequences obtained were compared with proprietary or public databases (Quast et al.,

2012) and Greengenes (DeSantis et al., 2006) containing several previously

characterized DNA sequences.

2.4. Beverage fermentation and drying

With the established fermentation conditions, the beverage was produced to be

dehydrated. For spray drying and lyophilization, three formulations with different types

of carriers were developed to evaluate the effect on microbial viability and the effect of

the obtained powders on the physicochemical characteristics. The feed solution T01

consisted of using 20% (w/v) of maltodextrin as a carrier, feed solution T02 was

composed of 10% (w/v) maltodextrin and 10% (w/v) inulin, and finally, the T03 feed
 10

solution had 20% (w/v) inulin as a carrier. The total solid contents were 17.65 ± 0.12

(T01), 17.44 ± 0.22 (T02), and 17.51 ± 0.22 (T03). The carriers were mixed with the

fermented beverage with a controllable speed homogenizer before and during drying.

2.4.1. Spray drying

Spray drying was performed through atomization in a laboratory-scale spray

dryer (B-290 mini spray dryer, Buchi, Flawil, Switzerland) at a constant inlet air

temperature of 135°C ± 1°C and a feed rate of 12 mL/min as per conditions described

by Atalar and Dervisoglu (2015) and Verruck et al. (2019), with adaptations. Under

these conditions, the outlet temperature was 40 ± 3°C.

2.4.2. Lyophilization

The different feed solutions (T01, T02, and T03) were frozen (-80 °C ± 2 for 24

h) in aluminum supports with a capacity of approximately 150 g of sample each on the

same day they were produced. Then, the samples were lyophilized in a Liotop L101

lyophilizer (São Carlos, São Paulo, Brazil) and removed from the lyophilizer 24 h later

(vacuum: 0.200–0.300 μHg and condenser temperature of -37 °C ± 2).

All lyophilized and spray-dried samples were submitted to physical-chemical

(pH, moisture, and water activity), physical (flow properties, hygroscopicity, solubility,

FTIR, DSC, color parameters, and particle morphology), and microbiological (lactic

acid bacteria, acetic bacteria, and molds and yeasts).

2.5. Microbiological analyzes

The serial dilutions of the feed solutions and the powdered kefir samples were

carried out by transferring 1mL to 9 mL of 0.1% peptone water (w/v) with subsequent
 11

inoculation in specific culture media. MRS agar was used to count lactic acid bacteria

(Merck, Germany), and the plates were incubated at 37°C under anaerobic conditions

for 72 h. DRBC agar was used to count molds and yeasts (Merck, Germany) with

incubation of the plates at 25°C for 24-72 h. For the identification of acetic bacteria,

Frateur agar (10 g/L yeast extract, 20 g/L Agar, 20 g/L calcium carbonate, and 20 g/L

ethanol) was used at an incubation temperature of 25°C for 5-10 days (Cassoni, 2008).

The results were expressed in colony-forming units per milliliter (CFU/mL).

2.6. Physicochemical analysis

The pH analysis was performed using the potentiometric method, introducing

the electrode directly into the samples and into the powders in a 1% solution (Mettler

Toledo Seven Easy S20, Ohio, United States). Moisture was determined by direct

heating in an oven at 105°C. Water activity was determined using an Aqualab® 3TE

digital hygrometer (Decagon Devices Inc, Pullman, USA) at 25 °C. All analyses

followed recommendations from the Adolfo Lutz Institute (2008).

2.7. Flow Properties

The bulk (ρbulk) and tapped (ρtapped) densities were calculated as proposed by

Jinapong et al. (2008), with modifications. For bulk density calculation, 2 g of each

spray-dried and lyophilized powder sample was gently placed in 10 mL graduated

cylinders, and the filled volume was read. The same procedure was performed for

tapped density (ρtapped). However, before reading the volume, the test tube was tapped

vigorously against a surface until there was no further change in volume. Both the bulk

and tapped density were calculated by dividing the mass of the dry powder by the

volume occupied in the graduated cylinder.

 12

2.8. Water Solubility

Water solubility was assessed by adding 1 g of each spray-dried and lyophilized

powder sample to 25 mL of distilled and stirred water for 5 min using a magnetic stirrer.

The solution was centrifuged at 760 × g for 10 min, and 20 mL aliquot of the

supernatant was transferred to a pre-weighed Petri dish and subjected to drying in an

oven at 105°C overnight, as described by Botrel et al. (2014). Water solubility (%) was

calculated as the percentage of dry supernatant compared to the initial amount of

powder (1g).

2.9. Fourier Transform Infrared

Fourier Transform Infrared obtained absorption spectra in the infrared region –

FTIR using the Perkin Elmer Spectrum 100 spectrophotometer with a resolution of

4/cm in the region of 4,000–600/cm with the attenuated total reflection technique (ATR)

with a germanium crystal.

2.10. Differential Scanning Calorimetry

The samples' differential scanning calorimetry (DSC) was obtained using a

Shimadzu DSC-50 (Shimadzu, Kyoto, Japan). Approximately 5mg of each sample was

placed in sealed aluminum containers under a dynamic synthetic air atmosphere of 20

mL min−1 and heated from −30 °C to 210 °C, with a heating rate of 5 °C min−1. The

DSC device was preliminarily calibrated with an indium reference standard (Verruck et

al., 2018).

2.11. Color Properties

 13

The color evaluation of the powders obtained was performed using a Byk

Gardner colorimeter (Konica Minolta), operating the CIELAB system, in which L*

(lightness), a* (red-green), and b* (yellow-blue) are the coordinates of chromaticity.

The total color difference (ΔE) between samples submitted to different methods was

calculated according to Verruck et al. (2015).

2.12. Hygroscopicity

The hygroscopicity of the probiotic microcapsules was determined according to

the method described by Dias et al. (2018). One gram of kefir powders was placed in

Petri dishes and then stored in a glass desiccator containing a saturated sodium chloride

solution (75.3% RH at 25 ± 1 °C) for seven days. Hygroscopicity was expressed in g of

moisture adsorbed per 100g of dry solids (g/100g).

2.13. Particle Morphology

Morphology was evaluated using a Jeol scanning electron microscope, model

JSM 6390 LV (Jeol, Tokyo, Japan), at an accelerating voltage of 10 kV. Samples were

fixed onto stubs and coated with gold using a cathodic vacuum atomization coater

(Leica, EM SCD 500, Wetzlar, Germany).

2.14. Statistical analysis

Fermentation kinetics, spray and lyophilization (freeze drying) experiments, and

powder characterization analyses were duplicated. The results were subjected to

analysis of variance and compared by Tukey's test at a significance level of 5% using

the Statistica software version 13.3 (TIBCO Software Inc., Palo Alto, CA, USA) with

the results expressed as mean ± standard deviation.

 14

3. Results and discussion

3.1. Fermentation Kinetics

Table 1 shows the values obtained for the growth of lactic acid bacteria,

substrate consumption, and mass growth of water kefir grains in different fermentation

conditions. It was observed that the counts of lactic acid bacteria in the conditions

evaluated ranged from 5.42  0.08 to 6.71  0.19 log CFU/mL. The lowest counts of

this group of microorganisms were attributed to the T4 and T8 experiments, which may

be related to the percentage of kefir grains greater than the amount of substrate

available. Due to the dynamics of fermentation, the yeasts in the kefir grains hydrolyze

sucrose through the invertase enzyme, thus raising the levels of glucose and fructose in

the medium. Lactic acid bacteria can use these monosaccharides as a carbon source if

the medium favors their growth (Fiorda et al., 2017). The rapid consumption of sucrose

in the T4 and T8 tests, with values between 73.05 ± 0.03 and 72.71 ± 0.01%, suggests

that yeast metabolism has been favored. According to Magalhães et al. (2010), during

the first 24 hours of fermentation, sucrose consumption is correlated with ethanol

generation by yeasts (mainly Saccharomyces cerevisiae).

Table 1. Response to different water kefir fermentation conditions

Independent Variables Response Variables

Substrate Lactic bacteria
Kefir Substrate Grain mass
Treatment (brown count (log CFU/
grains (%) consumption (%) growth (%)
sugar) (%) mL)
T1 5 5 6.50 ± 0.13ab 64.57 ± 0.03ab 74.10 ± 7.25ab
bc
T2 10 10 6.30 ± 0.13 67.06 ± 0.01ab 69.87 ± 1.76ab
T3 5 10 6.71 ± 0.19a 65.22 ± 0.01ab 100.86 ± 10.20a
e
T4 10 5 5.42 ± 0.08 73.05 ± 0.03a 54.64 ± 1.47b
cd
T5 5 5 6.14 ± 0.09 55.77 ± 0.02c 84.81 ± 7.34ab
T6 10 10 5.97 ± 0.17d 51.28 ± 0.03c 72.67 ± 7.54ab
 15

T7 5 10 6.29 ± 0.02b 51.73 ± 0.03c 96.91 ± 1.15ab

T8 10 5 5.52 ± 0.02e 72.71 ± 0.01a 57.26 ± 3.75b
Results are expressed as mean ± standard deviation (n = 2). Equal lowercase letters in the same column do not present
statistical differences among the conditions evaluated by Tukey's test (p>0.05). T1, T2, T3, and T4: incubation at 25
°C; T5, T6, T7, and T8: incubation at 30 °C.

The tests with the highest lactic acid bacteria counts were the T1 and T3 tests,

with values of 6.50 and 6.71 log CFU/mL, respectively. Under these conditions, the

proportions of available sucrose were 1:1 and 2:1 (sucrose: grains). The greater

substrate available in the T3 test hypothetically did not cause disorder in the common

fermentation dynamics. After the fermentation process, a statistically significant

difference (p<0.05) was found in the average increase in the masses of the kefir grains,

which showed a growth ranging from 54.64 ± 1.47% to 100.86 ± 10.20% compared to

the initial masses of the grains during fermentation. The increase in grain mass occurs

during the backslopping process (inoculum reuse for the next fermentation) and is

highly affected by the intrinsic characteristics of the grain, by environmental factors

such as available nutrients, grain inoculum, temperature, and fermentation time

(Laureys et al., 2019; Laureys and De Vuyst, 2017). The test with the highest grain

mass growth was the T3 test, in which substrate availability exceeds the number of kefir

grains inoculated. The same result was described by Laureys et al. (2018), in which the

condition with the highest concentration of nutrients improved grain growth.

Water kefir grain growth can slow down due to excess acid stress during

fermentation. Glucansucrases are enzymes lactic acid bacteria produce and are

responsible for water kefir grain growth. They are extracellular enzymes whose activity

is optimal at pH 4.0-5.5 and decreases at lower pH values (Côté and Skory, 2012;

Laureys et al., 2019; Waldherr et al., 2010). In this sense, the results shown in Figure 1

confirm that the T3 test at the end of 48 hours of fermentation presented the ideal pH

condition, with a value of 4.50.

 16

Figure 1 shows the pH variation curves for the different treatments and the

adjusted equations over time. The model proposed presented a good fit, i.e., good

agreement between the experimental values and the curve simulated by the model for all

treatments, as indicated by the high values of the coefficient of determination (R2)

(above 0.99). The evaluation of the pH value during fermentation proved to be a quick

and easy-to-obtain response, suitable for evaluating the fermentation process, regardless

of the processing conditions (incubation temperature and percentage of substrate).

Figure 1. Kinetics of pH variation for different water kefir fermentation conditions and
adjusted equations
 17

T1: 5% kefir grains, 5% brown sugar, and incubation at 25°C; T2: 10% kefir grains, 10% brown sugar, and
incubation at 25°C; T3: 5% kefir grains, 10% brown sugar, and incubation at 25°C; T4: 10% kefir grains, 5% brown
sugar, and incubation at 25°C; T5: 5% kefir grains, 5% brown sugar, and incubation at 30°C; T6: 10% kefir grains,
10% brown sugar, and incubation at 30°C; T7: 5% kefir grains, 10% brown sugar, and incubation at 30°C; T8: 10%
kefir grains, 5% brown sugar, and incubation at 30°C.
 18

According to Figure 1, the pH value of all evaluated conditions reduced over the

48 hours of fermentation. In general, it was observed that initially, the acidification

curve has a steeper slope between 5 and 15 hours of fermentation when the pH drops

more quickly. After this period, this slope becomes less pronounced, and the pH

decreases with less speed until it tends to remain in a range of very close values.

Experiments T5, T6, T7, and T8 that were incubated at 30°C showed the lowest pH

values at the end of 48 hours, all with values below 3.98 (Figure 1). Of these, the lowest

pH value observed was 3.94 for the T7 test. Velez and Peláez (2015) state that higher

incubation temperatures provide lower pH values, as observed in this study. In addition,

according to Montanuci (2010), the final pH of kefir fermentation is influenced by the

amount of inoculum used. This author reports that in the proportions of 1:10 (grains:

milk), pH values between 3.6 and 3.8 were found, while in the proportions of 1:30 and

1:50 (grains: milk), pH values of 4.4 to 4.6 were found, respectively. The greater the

proportion of inoculated grains, the lower the pH values. This result corroborates the

values obtained in this study since the T4 and T8 experiments, which are the ones with

the highest proportion of grains inoculated 2:1 (grains: substrate), were the experiments

that presented the lowest pH values at their respective incubation temperatures (25 and

30°C). Zongo et al. (2020) in their study to evaluate beverages fermented by water kefir

grains, observed that the pH decreased from 6.53 to 4.07 after two days of fermentation

at room temperature (22ºC). The same authors state that the drop in the samples' pH

values is expected, relating to the metabolism of microorganisms, mainly bacteria and

yeasts, which produce ethanol, volatile compounds, and carbon dioxide in addition to

producing acids.

The pH of food is considered an indicator of food safety. When the pH is in the

acidity range of less than 4.5, the growth of the main microorganisms responsible for
 19

foodborne illness is inhibited (Silva et al., 2018). Considering the value of 4.50 as the

optimal pH value for the completion of fermentation, we notice that the evaluated

experiments took different times to reach this value. At the end of the 48 hours of

fermentation, the predefined time for this study, the experiment that presented the

optimal pH value was the T3 experiment. This way, considering the pre-established

criteria for choosing the best fermentation condition, the T3 experiment with 5% grains,

10% sugar, and an incubation temperature of 25°C was considered the best condition to

proceed with the drying tests.

3.2. Molecular microbial identification

As a result of the metataxonomic analysis performed on kefir grains and

fermented beverage, it was found that the bacterial compositions comprised nine genera

(Figure 2). Within these genera, nine distinct species were identified, and approximately

94.31% of the composition of the grains and 91.68% of the composition of the

fermented beverage were of Zymomonas mobilis (Figure 2A). Zymomonas mobilis is an

aerotolerant, rod-shaped Gram-negative anaerobe (Weir, 2016). These bacteria produce

high levels of ethanol, competing with the yeast Saccharomyces cerevisiae in terms of

yield, and are associated with traditional fermented beverages in tropical regions of

America, Africa, and Asia (Marsh et al., 2014; Weir, 2016). Patel et al. (2022) found

similar results in evaluating water kefir microbiota and flavor attributes. On the other

hand, Cao et al. (2019) conducted a study to evaluate the biodiversity of Chinese water

kefir grains. It showed a different result from this study because Z. mobilis was

responsible for only about 1% of the relative abundance. This variation may be related

to the natural selection that can occur over several generations for the same type of
 20

beverage. Therefore, it is vital for product characterization to know the distinct

microbiological characteristics of production in different world regions.

Another species that appears in greater abundance when compared to the others

identified is the Sporolactobacillus spathodeae, with an abundance of 3.00% in kefir

grains and 5.42% in the fermented beverage. This species has yet to be described in any

other kefir or fermented beverage study. It is a lactic acid, endospore-forming, Gram-

positive species isolated by Thamacharoensuk et al. (2015) from tree bark in Thailand.

Liquorilactobacillus satsumensis was also identified in lesser abundance (1.47%

in the grains and 0.62% in the fermented beverage). This species was also identified in

water kefir grains in the Brazilian states of Goiás, Bahia, and Distrito Federal (Miguel et

al., 2011), in water kefir grains obtained from different regions of Germany, and also

isolated from shochu (Gulitz et al., 2013), a traditional Japanese distillate made from

fermented rice, sweet potato, barley, and other starches (Endo and Okada, 2005). The

other species identified represent about 1.23% of the relative abundance in kefir grains

and 2.28% in the fermented beverage. Within these percentages are included in a

combined way (both grains and beverage) the genus Lactobacillus sp. (1.35%) and the

species Acetobacter peroxydans (0.74%), Lentilactobacillus hilgardii (0.46%),

Oenococcus kitaharae (0.54%), Gluconobacter frateurii (0.23%), and Leuconostoc

mesenteroides (0.19%).
 21

Figure 2. Relative abundance (%) of bacteria (A) and fungi (B) species in water kefir

grains and the fermented beverage

Fermented Beverage

Grain
A
0% 20% 40% 60% 80% 100%
Relative abundance of bacterial species (%)
Zymo monas mobilis Sporolactobacillus spathodeae Liquorilactobacillus satsumensis
Lactobacillus sp. Lentilactobacillus hilgardii Oenococcus kitaharae
Acetobacter peroxydans Gluconobacter frateurii Leuconostoc mesenteroides

Fermented Beverage

Grain
B
0% 20% 40% 60% 80% 100%
Relative abundance of fungal species (%)

Lachancea fermentati Wicke rh amomyces anomalus Saccharomycetes sp.

Saccharomyces cerevisiae Torulaspora delbrueckii Candida californica

Concerning fungal diversity, all readings were assigned to the phylum

Ascomycota. At the family level, Saccharomycetaceae, Pichiacea, and

Phaffomycetaceae were detected in the grains and fermented beverages. Five genera

were identified in the grains and fermented beverages (Figure 2). The species

Lachancea fermentati corresponded to 95.54% of the composition of the grains and

67.53% of the composition of the fermented beverage (Figure 2B). For years, this
 22

species has been associated with grape must and wine fermentation processes in several

wine-producing countries (Porter et al., 2019). Unlike what was obtained in this study,

Gulitz et al. (2011) and Cordeiro (2018) found lower relative abundances for the genus

L. fermentati. The percentage was 6-8% in fermented beverages based on water kefir

and 2.27% in kefir grains obtained from consumers in the Brazilian state of Paraná,

respectively.

The second-highest relative abundance was registered for Wickerhamomyces

anomalus, with 3.00% in the grain and 26.77% in the fermented beverage. W.

anomalus, formerly known as Hansenula anomala, occur naturally in grape must and

has been evaluated for enhancing the aroma of wines by producing volatile compounds

(Lombard, 2016). Laureys et al. (2017) also identified the species W. anomalus while

investigating water kefir grains' instability and low growth during an industrial

fermentation process. The same identification occurred in the study of isolation and

molecular identification of yeasts in kefir, albeit in milk kefir grains obtained through

artisanal producers in Greece (Kalamaki and Angelidis, 2017).

Fungal species with lesser representation were also identified, such as the genus

Saccharomycetes sp. (0.66% in the grain and 0.60% in the fermented beverage) and the

species Saccharomyces cerevisiae (0.55% in the grain and 4.03% in the fermented

beverage), Torulaspora delbrueckii (0.14% in the grain and 0.33% in the fermented

beverage), and Candida californica (0.11% in the grain and 0.73% in the fermented

beverage).

3.3. Microbiological analysis of dehydrated water kefir

Table 2 presents the results of microbial counts demonstrating the effect of the

three compositions of carrier materials and two drying methods for the powders
 23

obtained from the best fermentation condition previously evaluated for water kefir. The

lactic acid bacteria counts in the different feed solutions did not differ significantly from

each other (p>0.05). When these solutions were subjected to spray drying, there was a

reduction (p<0.05) in the initial count. Thermal, osmotic, oxidative, and desiccation

stresses are generally considered the main mechanisms that cause bacterial inactivation

during and after spray drying (Arepally et al., 2020). Cellular damage caused by heat

action includes DNA and RNA denaturation, ribosomal damage, dehydration, and

plasma membrane destabilization due to water removal (Cebrián et al., 2017). Teijeiro

et al. (2018) reported that after spray drying, the viability of lactic acid bacteria in kefir

powder made with UHT milk was reduced by approximately 2.5 logs CFU/g.

Table 2. Microbial counts before and after spray drying and lyophilization
Lactic acid bacteria (log Molds and Yeasts (log Acetic acid bacteria (log
Test
CFU/g) CFU/g) CFU/g)
FST01 7.17 ± 0.43aA 5.14 ± 0.15aA 5.04 ± 0.22aA
FST02 7.07 ± 0.25aA 5.65 ± 0.42aA 5.25 ± 0.65aA
FST03 7.19 ± 0.55aA 5.53 ± 0.40aA 5.75 ± 0.37aA
T01S 3.76 ± 0.20aB 2.99 ± 0.21aB 2.38 ± 0.58aB
T02S 3.41 ± 0.48aB 3.03 ± 0.29aB 2.69 ± 0.53aB
T03S 3.85 ± 0.36aB 3.19 ± 0.43aB 2.92 ± 0.67aB
T01F 6.35 ± 0.18aA 5.03 ± 0.08aA 4.80 ± 0.02aA
T02F 6.75 ± 0.15aA 5.40 ± 0.01aA 4.46 ± 0.30aA
T03F 6.69 ± 0.83aA 5.41 ± 0.03aA 4.66 ± 0.26aA
Results are expressed as mean ± standard deviation (n = 2). Different lowercase letters between lines show a
significant difference (p<0.05) among the evaluated tests. Different capital letters between lines are significantly
different (p<0.05) between the drying processes used. FS – Feed solution before the drying process; T01S – test using
100% maltodextrin as dry carrier in spray dryer; T02S – test using 50% maltodextrin and 50% inulin as dry carriers
in spray dryer; T03S – test using 100% inulin as dry carrier in spray dryer; T01F – test using 100% maltodextrin as
dry carrier by lyophilization; T02F – test using 50% maltodextrin and 50% inulin as dry carriers by lyophilization;
T03F – test using 100% inulin as lyophilized carrier.

No differences were shown (p>0.05) for the mold and yeast counts in the

different feed solutions. This result corroborates the one obtained by Rodríguez et al.

(2023), who found yeast counts close to 5.30 logs CFU/g, and with the study of

Villanoeva et al. (2021), who reported a value of 5.89 to 6.86 log CFU/g of yeasts for

water kefir. In optimizing spray drying parameters of kefir powder, Atalar and
 24

Dervisoglu (2015) found yeast counts in the 4.54–5.89 logs CFU/mL range. In our

study, spray drying promoted a logarithmic reduction (p<0.05) of 2.15, 2.62, and 2.34

for T01, T02, and T03, respectively. This result demonstrates that spray drying failed to

maintain the viability of molds and yeasts. The low thermotolerance of yeasts has been

reported because temperatures above 50 °C markedly decrease their survival (Tontul et

al., 2021). When the feed solutions were subjected to the lyophilization process, no

difference (p>0.05) was observed in the reduction of viability of these microorganisms.

In this sense, lyophilization emerges as the best drying technique for water kefir, which

aligns with the conclusions obtained in investigations focused on drying milk kefir

(Teijeiro et al., 2018).

Similar behavior was observed regarding acetic bacteria when the T01, T02, and

T03 were submitted to the drying processes. Thus, there was a reduction in the initial

viability for tests dried in a spray dryer and maintenance of viability for tests submitted

to lyophilization (Table 1). Tontul et al. (2021) obtained similar results; however, they

informed that it is difficult to obtain linearity of the results since there are differences in

the tolerance of microorganisms.

The thermoprotective effect of some carriers in spray drying and lyophilization

is being evaluated. Usually, the carriers help maintain the viability of microorganisms

(Coutinho Favilla et al., 2022; Teijeiro et al., 2018). Within the scope of this research,

the evaluated wall materials (maltodextrin and inulin) showed the same performance. In

this way, choosing any carrier tested for drying water kefir becomes possible. Another

point observed is that the spray dryer parameters used in this study, according to the

optimization performed by Atalar and Dervisoglu (2015), were not ideal, considering

the evaluated microorganisms' viability loss. There are many manners to optimize the

drying process using a spray dryer. In addition to using carriers and changing the inlet
 25

temperature, adaptations can be made to the spray dryer equipment, such as Tabatabaei

et al. (2022), which utilized a spray dryer equipped with an electrostatic collector to dry

chitosan. This new technology can be used in future studies to verify the protective

effect during the drying of liquid foods containing microorganisms.

3.4. Physical-chemical analyses of the powders

The physical characteristics of spray-dried and lyophilized powders made with

different carriers can vary according to the methods used in production and can impact

their functionality. Therefore, Table 3 presents the results of the physicochemical

properties evaluated.

The lowest (p<0.05) pH value was attributed to T01 considering spray drying,

while for lyophilization, the sample that presented the highest (p<0.05) value was T03.

When comparing the drying methods, the samples submitted to the lyophilization

process showed higher pH values (p<0.05) than those submitted to spray drying. During

lyophilization, as water is removed from the material, the concentration of ions in the

solution can increase, which may increase pH (Challener, 2017).

Table 3. Results expressed as mean ± standard deviation of the physicochemical

properties of the spray-dried and lyophilized powders

Analysis Test Spray dried powder Lyophilized powder

T01 5.24± 0.02bB 5.35 ± 0.05bA
pH T02 5.32 ± 0.02aB 5.40 ± 0.02bA
T03 5.32 ± 0.01aB 5.52 ± 0.04aA
T01 0.242 ± 0.004bB 0.336 ± 0.002bA
Water activity T02 0.254 ± 0.011bB 0.341 ± 0.023bA
T03 0.371 ± 0.005aA 0.392 ± 0.024aA
T01 3.05 ± 0.03abB 4.42 ± 0.01aA
Moisture (%) T02 3.19 ± 0.12aB 3.73 ± 0.06bA
T03 2.68 ± 0.27bB 3.62 ± 0.05bA
T01 12.89 ± 1.65bA 12.28 ± 1.55bA
Hygroscopicity (%)
T02 16.04 ± 0.35aB 16.91 ± 0.38aA
 26

T03 16.41 ± 0.28aB 17.44 ± 0.45aA

T01 79.09 ± 1.18aA 75.05 ± 1.72aB
Solubility (%) T02 81.34 ± 1.66aA 70.93 ± 3.33aB
T03 81.26 ± 1.99aA 72.87 ± 1.63aB
T01 0.326 ± 0.026bA 0.382 ± 0.025bA
Bulk density (g/cm3) T02 0.374 ± 0.011bA 0.346 ± 0.048bA
T03 0.565 ± 0.018aA 0.557 ± 0.02aA
T01 0.448 ± 0.006bA 0.360 ± 0.021bB
Tapped density (g/cm3) T02 0.592 ± 0.007aA 0.391 ± 0.066aB
T03 0.636 ± 0.010aA 0.410 ± 0.011aB
T01 85.17 ± 0.01aB 86.94 ± 0.49aA
L* T02 84.62 ± 0.08aB 87.88 ± 0.12aA
T03 84.56 ± 0.08aB 86.50 ± 0.40aA
T01 1.62 ± 0.01aA 1.61 ± 0.07aA
a* T02 1.62 ± 0.09aA 1.61 ± 0.05aA
T03 1.61 ± 0.04aA 1.61 ± 0.03aA
T01 10.55 ± 0.03aB 12.97 ± 0.05aA
b* T02 10.72 ± 0.06aB 12.31 ± 0.32aA
T03 10.47 ± 0.08aB 13.36 ± 0.16aA
T01 3.01
∆E* T02 3.63
T03 3.49

Results are expressed as mean ± standard deviation (n = 3). Different lowercase letters between lines show a
significant difference (p<0.05) between the different wall materials. Values followed by different capital letters
between columns differ significantly (p<0.05) between drying processes. T01 – test using 100% maltodextrin as the
carrier; T02 – test using 50% maltodextrin and 50% inulin as the carrier; T03 – test using 100% inulin as the carrier.
L* indicates the lightness, a* the variation from red (+a*) to green (–a*), b* the variation from yellow (+b*) to blue
(–b*), and ΔE total color difference.

The food industry knows that powdered products' ideal moisture content should

be between 4 and 7% so that they are stable during storage (Teijeiro et al., 2018). Thus,

all powders obtained meet this premise and are considered stable products. In addition,

moisture control is crucial as it strongly impacts microbial viability during storage,

which is one of the quality parameters for powders containing viable microorganisms

(Sakoui et al., 2022).

Water activity (aw) is related to the availability of free water in the sample and is

also an important parameter that determines powders' stability and shelf life during

storage (Wang et al., 2020). In the present study, aw showed behavior similar to the

moisture analysis results. According to Tapia et al. (2020), water activity values

between 0.20 and 0.40 guarantee the stability of the stored product against browning
 27

and hydrolytic reactions, liquid oxidation, auto-oxidation, and enzymatic activity.

Therefore, all the powders obtained in the present study are within the range considered

optimal for the stability of powdered products.

Powdered foods with less than 10% hygroscopicity are classified as non-

hygroscopic (Costa et al., 2014). Therefore, the kefir powders in the present study

obtained through spray drying and lyophilization are classified as hygroscopic with

potential impairment of shelf-life, demanding storage in a controlled environment and

special packaging. The powder produced only with maltodextrin (T01) was the powder

with the lowest (p<0.05) hygroscopic nature for both drying processes. As the addition

of inulin was performed as wall material in T02 and T03, an increase in hygroscopicity

was observed. This behavior differs from what Mensink et al. (2015) expect. According

to the authors, inulin is a material with a higher degree of polymerization, has fewer

reducing groups, has a higher glass transition temperature, and, therefore, has a lower

tendency to absorb moisture from the environment.

Solubility is extremely important for powdered products, and high solubility is

desirable (Bicudo et al., 2015). Different factors such as the chemical composition of

food (surface composition, hydrophobicity, and others), physical properties (particle

size, porosity, shape, and others), and wall materials (if any) affect the solubility of

powdered food (Tontul and Topuz, 2017). In the present study, the type of wall material

used had no significant effect on powder solubility, while the drying method applied

had a significant influence (p<0.05). The tests submitted to the spray drying process

showed solubility greater (p<0.05) than the solubility of the lyophilized samples. The

higher solubility of the samples submitted to the spray drying process may be due to the

smaller particle size obtained for these samples since the smaller the particle size, the

greater the surface area available for hydration (Kuck and Noreña, 2016).
 28

In addition to hygroscopicity and solubility, measuring physical properties such

as density is essential because they inherently affect the behavior of the powder during

storage, handling, transport, processing, and packaging (Verruck et al., 2018).

Regarding density, it was observed that the carrier agents employed influenced and

caused differences between the values obtained for the tapped and bulk densities of the

powders. T03 subjected to spray drying and lyophilization for bulk density showed

higher values (p<0.05) than the others. Lower bulk densities result in a larger package

volume; therefore, they are undesired. On the other hand, high bulk densities increase

powder stability and improve protection against oxidation (Himmetagaoglu and Erbay,

2019).

Furthermore, it was observed that the tapped density of the powders tended to

increase as inulin was added to the feed solution as wall material. That way, the

powders obtained for T02 and T03 through spray drying and lyophilization were equal

(p>0.05) to each other and greater than those obtained in test T01 with 100%

maltodextrin as a carrier agent. Regardless, when comparing the drying methods, the

powders obtained by lyophilization showed lower (p<0.05) tapped density than spray

drying. The lower tapped density of samples produced by lyophilization may be related

to the more porous structure caused by the drying method, thus causing a high volume

of air in the structure (Preethi et al., 2021).

For the color parameters, despite varying (p<0.05) between the drying methods,

it is noted that all powders had high L* values, which indicates a light color with a

tendency towards white. The parameter a* indicates a predominance of reddish

coloration, while the b* parameter suggests a yellowish color, which can be attributed to

the brown sugar in the test formulation. No difference (p>0.05) was observed between

the drying methods used for the parameter a*, suggesting that this parameter low
 29

influences the color of the sample differences observed in ΔE*. Thus, Martínez-

Cervera et al. (2011) report that the human eye cannot visually perceive total color

difference (ΔE*) values smaller than 3. Thus, it was possible to verify that all evaluated

samples were affected by the drying methods, suggesting a significant change in color

perception. Therefore, monitoring ΔE* is crucial to assess product quality and

acceptability, ensuring that color variations do not compromise the sensory experience

or consumer acceptance.

3.5. Fourier Transform Infrared spectroscopy

FTIR analysis can be applied to investigate the chemical and structural changes

that occur during spray drying and lyophilization of water kefir, using different carriers,

such as inulin, maltodextrin, and the mixture of the two materials. Figure 3 (I) shows

the FTIR spectra for spray-dried and lyophilized kefir powders. When analyzing the

spectra, the most significant absorption bands, which provide more information about

samples, range from 3409 to 2889/cm and from 1647 to 930/cm (fingerprint region). It

is important to emphasize that these bands are present in all samples.

At 3409/cm, a broad and intense band is observed, which refers to stretching-H

bonds in carbohydrate molecules, such as maltodextrin and inulin (Barbosa, 2013; Paiva

et al., 2013). At 2925 and 2893/cm, two bands of medium intensity are observed,

referring to asymmetric and symmetric stretching of C-H bonds of sp³ carbons (Barbosa,

2013; Paiva et al., 2013). In addition to these bands, there is also a band at 1648/cm,

which is related to the stretching of the C=O bond of carboxylic acids and esters,

possibly formed in fermentation processes (Paiva et al., 2013; Romano et al., 2018).

Finally, at 1080, 1028, 999, and 930/cm, intense bands are observed inherent to the

stretching of ether groups present in carbohydrates and of the O-C-O glycosidic bonds
 30

of saccharides, such as maltodextrin and inulin (Barbosa, 2013; Paiva et al., 2013;

Romano et al., 2018). These observations indicate that the carriers used (inulin,

maltodextrin, and their mixture) do not significantly alter the fundamental chemical

structure of the water kefir, as evidenced by the consistent presence of these

characteristic absorption bands across all samples. The data suggest that both spray

drying and lyophilization processes maintain the integrity of the kefir's key chemical

components when these carriers are used.

Figure 3. Fourier transform infrared (FTIR) spectroscopy (I), scanning electron

microscopy (SEM), and thermograms (III) of spray-dried and lyophilized water kefir

powders

T01S – test using 100% maltodextrin as carrier in spray dryer; T02S – test using 50% maltodextrin and 50% inulin as
carriers in spray dryer; T03S – test using 100% inulin as carrier in spray dryer; T01F – test using 100% maltodextrin
as carrier in lyophilization; T02F – test using 50% maltodextrin and 50% inulin as carriers in lyophilization; T03F –
test using 100% inulin as carrier in lyophilization. A, B, and C: spray-dried kefir particles using 100% maltodextrin,
50% maltodextrin, and 50% inulin; and 100% inulin, respectively; D, E, and F: lyophilized kefir particles using 100%
maltodextrin, 50% maltodextrin, and 50% inulin; and 100% inulin, respectively.

3.6. Morphology by Scanning Electron Microscopy

 31

Figure 3 (II) shows the micrographs obtained by Scanning Electron Microscope

– SEM of the kefir powder particles produced using different carrier agents and drying

methods. The kefir powder particles produced by lyophilization were perceived as

porous, irregularly shaped, and disordered. Ice crystals form due to the low

temperatures and sublimate under reduced pressure, forming dry and porous products

(Dolly et al., 2011). Cavalcante (2016) observed similar morphology in soursop pulp

particles obtained through lyophilization. Caparino et al. (2012) also observed porous

surfaces when analyzing microscopic images of powdered mango pulp obtained through

lyophilization. On the other hand, the spray-dried kefir microparticles showed different

morphologies compared to the lyophilized samples and differences concerning the

carrier agents used.

For the sample produced using maltodextrin as wall material (Fig. 3 (II) A), the

microcapsules were spherical, smooth surfaces and without cracks or pores. As inulin

was incorporated into the samples as a wall material, the microcapsules showed ovoid

morphology and apparent roughness (Fig. 3 (II) B and C). Another point observed is

that with the inclusion of inulin in the samples, the particles increased in size, as shown

in the images (Fig. 3 (II) B and C). This increase corroborates the previously mentioned

density values since, as inulin was included as a carrier agent, the values of tapped and

bulk densities also increased. Silva (2021) found similar behavior when maltodextrin,

inulin, and the combination of both were evaluated as carrier agents in the drying

process of sapota-do-solimoes extract (Matisia cordata). The samples in which inulin

was used as a carrier also had a larger particle size.

3.7. Differential Scanning Calorimetry

 32

DSC analysis can be applied to investigate the thermal changes in water kefir

during spray drying and lyophilization using different carriers, such as inulin,

maltodextrin, and a mixture of the two materials. The DSC curves of the evaluated

samples are shown in Figure 3 (III). The curve belonging to the fermented beverage

showed some distinct endothermic events between 65 and 200 °C. For this event, the

temperature peak at 114.25 °C stands out, which is characteristic of the evaporation of

the water present in the beverage formulation. Another observed peak occurred at a

temperature of 179.75°C (ΔH = 24.0024 J/g), which may be associated with the melting

and caramelization of sucrose, a constituent of brown sugar used as a substrate in the

beverage's fermentation. In the literature, there are quite variable melting temperature

records for sucrose with values of 165, 176, 185.9, 184.1, and 192 °C (Maia, 2019;

Wang et al., 2019).

The DSC curves of the spray-dried kefir powders showed a well-defined thermal

event at temperatures of 190.65, 195.15, and 198.82 °C relative to tests T01S, T02S,

and T03S, respectively. The endothermic peaks observed in the tests are related to the

loss of hydrophilic groups present in the sample's characteristic of their melting point.

The calculated enthalpy of fusion (ΔH) was 43.9302, 26.0924, and 1.2326 J/g relative to

tests T01S, T02S, and T03S, respectively. For the samples obtained by the

lyophilization method, the characteristic peaks of powder melting were observed at

temperatures of 194.73, 176.56, and 200.46 °C (ΔH = 11.5670, 3.1191, and 1.6036 J/g)

for tests T01F, T02F, and T03F, respectively.

The endothermic peaks observed in the T01S and T01F tests (194.65 and 194.73

°C) that contain maltodextrin as a carrier suggest a better efficiency in the encapsulation

process when compared to the other tests. This hypothesis can be inferred from the

difference in values of the characteristic endothermic peaks at the melting point of the
 33

tests mentioned and pure maltodextrin (241.05 °C; ΔH = 95.9417 J/g). According to

Maia (2019), when microcapsule formation does not occur, the endo and exothermic

peaks remain close to the peak values of pure substances.

It is also verified that as inulin is added as a carrier in the T02S, T03S, T02F,

and T03F tests, there is an increase in the endothermic temperature when compared to

the T01S and T01F tests in which 100% maltodextrin was used as a carrier. This

behavior suggests a lower efficiency in the encapsulation process since the peak

temperatures of the T02S, T03S, T02F, and T03F tests are close to — or at least

approaching — the temperature characteristic of the inulin melting process (203.48 °C;

ΔH = 28.2166 J/g). Based on these results, it can be stated that the microencapsulation

process using maltodextrin presents remarkable efficiency and, therefore, a tendency

towards stability of the powders obtained compared to the tests using inulin as a carrier.

4. Conclusions and Recommendations

The physical and physicochemical properties evaluated revealed that water kefir

dehydration is a feasible alternative to implement in the beverage industry. The optimal

fermentation conditions were achieved using 5% water kefir grains, 10% sugar, and an

incubation temperature of 25°C. Considering the potentiality of offering a source of

probiotics and viable microorganisms, drying kefir by lyophilization was the best

method for producing stable food with preserved viability of the microorganisms

present in the beverage.

Based on the results obtained, some directions for future research on powdered

water kefir can be recommended. Firstly, evaluating the effects of more different types

of carriers, such as proteins or gums, on the microbial viability and physicochemical

 34

characteristics of water kefir powders dehydrated by spray drying and lyophilization

would be beneficial. Additionally, it is important to investigate how dehydration

impacts water kefir's functional and bioactive properties, including its antioxidant and

antimicrobial activities. Conducting accelerated stability studies to determine the shelf

life of water kefir powders and identifying key factors that affect their longevity would

also provide valuable insights. Furthermore, developing reconstituted beverage

formulations from water kefir powders and assessing their sensory acceptance and

consumer purchase intent could enhance product development. Another promising area

of study is exploring the prebiotic potential of exopolysaccharides produced during

water kefir fermentation and their effects on gut microbiota modulation. Finally,

conducting in vivo studies in animals and humans to evaluate the health benefits of

regular water kefir consumption, such as improved immune function and reduced

inflammatory markers, would further substantiate the positive impacts of water kefir on

health.

CRediT authorship contribution statement

Klinger Vinícius de Almeida: Formal analysis, Conceptualization, Data curation,

Writing – original draft. Vanessa Cortina Zanetti: Formal analysis, Data curation.

Callebe Camelo Silva: Formal analysis, Data curation. Luan Alexandre Amaral:

Formal analysis. Alice Cristina da Silva: Formal analysis. Silvani Verruck:

Conceptualization, Formal analysis, Data curation, Validation, Writing – review &

editing, Funding acquisition, Project administration. Luciano José Quintão Teixeira:

Conceptualization, Data curation, Validation, Writing – review & editing, Funding

acquisition, Project administration.

 35

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or

personal relationships that could have appeared to influence the work reported in this

paper.

Acknowledgments

The authors would like to thank CNPq (National Council for Scientific and

Technological Development – process number 306063/2022-0 and 306063/2022-0) and

the Foundation for Research Support of Santa Catarina - FAPESC [grants number

2021TR001446 and 2022TR002005] for the financial support.

References
Alves, V., Scapini, T., Camargo, A. F., Bonatto, C., Stefanski, F. S., Pompeu de Jesus,
E., Techi Diniz, L. G., Bertan, L. C., Maldonado, R. R., & Treichel, H. (2021).
Development of fermented beverage with water kefir in water-soluble coconut
extract (Cocos nucifera L.) with inulin addition. LWT, 145, 111364.
https://doi.org/10.1016/j.lwt.2021.111364

Arepally, D., Reddy, R. S., & Goswami, T. K. (2020). Studies on survivability, storage
stability of encapsulated spray dried probiotic powder. Current Research in Food
Science, 3, 235–242. https://doi.org/10.1016/j.crfs.2020.09.001

Atalar, I., & Dervisoglu, M. (2015). Optimization of spray drying process parameters
for kefir powder using response surface methodology. LWT - Food Science and
Technology, 60(2), 751–757. https://doi.org/10.1016/j.lwt.2014.10.023

Barbosa, L. C. A. (2013). Espectroscopia no infravermelho na caracterização de

compostos orgânicos. (1a Edição). Editora UFV.

Bicudo, M. O. P., Jó, J., Oliveira, G. A. de, Chaimsohn, F. P., Sierakowski, M. R.,
Freitas, R. A. de, & Ribani, R. H. (2015). Microencapsulation of Juçara (Euterpe
edulis M.) Pulp by Spray Drying Using Different Carriers and Drying
Temperatures. Drying Technology, 33(2), 153–161.
https://doi.org/10.1080/07373937.2014.937872

Botrel, D. A., Borges, S. V., Fernandes, R. V. D. B., & Lourenço Do Carmo, E. (2014).
Optimization of Fish Oil Spray Drying Using a Protein:Inulin System. Drying
Technology, 32(3), 279–290. https://doi.org/10.1080/07373937.2013.823621
 36

Cao, C., Hou, Q., Hui, W., Kwok, L., Zhang, H., & Zhang, W. (2019). Assessment of
the microbial diversity of Chinese Tianshan tibicos by single molecule, real-time
sequencing technology. Food Science and Biotechnology, 28(1), 139–145.
https://doi.org/10.1007/s10068-018-0460-8

Caparino, O. A., Tang, J., Nindo, C. I., Sablani, S. S., Powers, J. R., & Fellman, J. K.
(2012). Effect of drying methods on the physical properties and microstructures of
mango (Philippine 'Carabao' var.) powder. Journal of Food Engineering, 111(1),
135–148. https://doi.org/10.1016/j.jfoodeng.2012.01.010

Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J., Fierer, N.,
Owens, S. M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J. A., Smith,
G., & Knight, R. (2012). Ultra-high-throughput microbial community analysis on
the Illumina HiSeq and MiSeq platforms. The ISME Journal, 6(8), 1621–1624.
https://doi.org/10.1038/ismej.2012.8

Caro Velez, C. A., & León Peláez, Á. M. (2015). Capacidad antifúngica de

sobrenadantes libres de células obtenidos de la fermentación de un sustrato de
“panela” con gránulos de kefir de agua. Revista Colombiana de Biotecnología,
17(2), 22–32. https://doi.org/10.15446/rev.colomb.biote.v17n2.42758

Cassoni, V. (2008). Valorização de resíduo de processamento de farinha de mandioca

(Manipueira) por acetificação [Dissertação - Mestrado em Agronomia].
Universidade Estadual Paulista “Julio de Mesquita Filho.”

Cavalcante, C. E. B. (2016). Secagem de polpa de graviola em spray-dryer e

liofilizador: Avaliação das características físico-químicas, higroscópicas e
morfológicas [Tese Doutorado em Ciência e Tecnologia de Alimentos].
Universidade Federal do Ceará.

Cebrián, G., Condón, S., & Mañas, P. (2017). Physiology of the inactivation of
vegetative bacteria by thermal treatments: Mode of action, influence of
environmental factors and inactivation kinetics. Foods, 6(12), 107.
https://doi.org/10.3390/foods6120107

Challener, C. A. (2017). For Lyophilization, Excipients Really Do Matter. BioPharm

International, 30, 32–35.

Chaturvedi, S., Khartad, A., & Chakraborty, S. (2021). The potential of non-dairy
synbiotic instant beverage powder: Review on a new generation of healthy ready-
to-reconstitute drinks. Food Bioscience, 42, 101195.
https://doi.org/10.1016/j.fbio.2021.101195

Cordeiro, N. A. O. (2018). Kefir de água BIONAT-I: análise metagenômica dos grãos,

obtenção e caracterização estrutural do exopolissacarídeo consorciana e seu
potencial biotecnológico. Universidade Federal de Pernambuco.

Corona, O., Randazzo, W., Miceli, A., Guarcello, R., Francesca, N., Erten, H.,
Moschetti, G., & Settanni, L. (2016). Characterization of kefir-like beverages
 37

produced from vegetable juices. LWT - Food Science and Technology, 66, 572–
581. https://doi.org/10.1016/j.lwt.2015.11.014

Costa, J. de P. da, Rocha, É. M. de F. F., & Costa, J. M. C. da. (2014). Study of the
physicochemical characteristics of soursop powder obtained by spray-drying. Food
Science and Technology (Campinas), 34(4), 663–666.
https://doi.org/10.1590/1678-457X.6380

Côté, G. L., & Skory, C. D. (2012). Cloning, expression, and characterization of an

insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL
B-1118. Applied Microbiology and Biotechnology, 93(6), 2387–2394.
https://doi.org/10.1007/s00253-011-3562-2

Coutinho Favilla, A. L., Rosa dos Santos Junior, E., Novo Leal Rodrigues, M. C.,
Baião, D. dos S., Flosi Paschoalin, V. M., Lemos Miguel, M. A., da Silva Carneiro,
C., & Trindade Rocha Pierucci, A. P. (2022). Microbial and physicochemical
properties of spray dried kefir microcapsules during storage. LWT, 154, 112710.
https://doi.org/10.1016/j.lwt.2021.112710

da C. P. Miguel, M. G., Cardoso, P. G., Magalhães, K. T., & Schwan, R. F. (2011).

Profile of microbial communities present in tibico (sugary kefir) grains from
different Brazilian States. World Journal of Microbiology and Biotechnology,
27(8), 1875–1884. https://doi.org/https://doi.org/10.1007/s11274-010-0646-6

da Silva Fernandes, M., Sanches Lima, F., Rodrigues, D., Handa, C., Guelfi, M., Garcia,
S., & Ida, E. I. (2017). Evaluation of the isoflavone and total phenolic contents of
kefir-fermented soymilk storage and after the in vitro digestive system simulation.
Food Chemistry, 229, 373–380. https://doi.org/10.1016/j.foodchem.2017.02.095

de Souza, H. F., Bogáz, L. T., Monteiro, G. F., Freire, E. N. S., Pereira, K. N., de
Carvalho, M. V., da Silva Rocha, R., da Cruz, A. G., Brandi, I. V., & Kamimura,
E. S. (2024). Water kefir in co-fermentation with Saccharomyces boulardii for the
development of a new probiotic mead. Food Science and Biotechnology.
https://doi.org/10.1007/s10068-024-01568-2

de Souza, H. F., Monteiro, G. F., Bogáz, L. T., Freire, E. N. S., Pereira, K. N., Vieira de
Carvalho, M., Gomes da Cruz, A., Viana Brandi, I., & Setsuko Kamimura, E.
(2024). Bibliometric analysis of water kefir and milk kefir in probiotic foods from
2013 to 2022: A critical review of recent applications and prospects. Food
Research International, 175, 113716.
https://doi.org/10.1016/j.foodres.2023.113716

DeSantis, T. Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E. L., Keller, K., Huber,
T., Dalevi, D., Hu, P., & Andersen, G. L. (2006). Greengenes, a Chimera-Checked
16S rRNA Gene Database and Workbench Compatible with ARB. Applied and
Environmental Microbiology, 72(7), 5069–5072.
https://doi.org/10.1128/AEM.03006-05

Dias, C. O., dos Santos Opuski de Almeida, J., Pinto, S. S., de Oliveira Santana, F. C.,
Verruck, S., Müller, C. M. O., Prudêncio, E. S., & de Mello Castanho Amboni, R.
 38

D. (2018). Development and physico-chemical characterization of

microencapsulated bifidobacteria in passion fruit juice: A functional non-dairy
product for probiotic delivery. Food Bioscience, 24, 26–36.
https://doi.org/10.1016/j.fbio.2018.05.006

Dolly, P., Anishaparvin, A., Joseph, G. S., & Anandharamakrishnan, C. (2011).

Microencapsulation of Lactobacillus plantarum (mtcc 5422) by spray-freeze-
drying method and evaluation of survival in simulated gastrointestinal conditions.
Journal of Microencapsulation, 28(6), 568–574.
https://doi.org/10.3109/02652048.2011.599435

Endo, A., & Okada, S. (2005). Lactobacillus satsumensis sp. nov., isolated from mashes
of shochu, a traditional Japanese distilled spirit made from fermented rice and
other starchy materials. International Journal of Systematic and Evolutionary
Microbiology, 55(1), 83–85. https://doi.org/10.1099/ijs.0.63248-0

Fiorda, F. A., de Melo Pereira, G. V., Thomaz-Soccol, V., Rakshit, S. K., Pagnoncelli,
M. G. B., Vandenberghe, L. P. de S., & Soccol, C. R. (2017). Microbiological,
biochemical, and functional aspects of sugary kefir fermentation - A review. Food
Microbiology, 66, 86–95. https://doi.org/10.1016/j.fm.2017.04.004

Gökırmaklı, Ç., Şatır, G., & Guzel‐ Seydim, Z. B. (2024). Microbial viability and
nutritional content of water kefir grains under different storage conditions. Food
Science & Nutrition. https://doi.org/10.1002/fsn3.4074

Gulitz, A., Stadie, J., Ehrmann, M. A., Ludwig, W., & Vogel, R. F. (2013).
Comparative phylobiomic analysis of the bacterial community of water kefir by
16S rRNA gene amplicon sequencing and ARDRA analysis. Journal of Applied
Microbiology, 114(4), 1082–1091. https://doi.org/10.1111/jam.12124

Gulitz, A., Stadie, J., Wenning, M., Ehrmann, M. A., & Vogel, R. F. (2011). The
microbial diversity of water kefir. International Journal of Food Microbiology,
151(3), 284–288. https://doi.org/10.1016/j.ijfoodmicro.2011.09.016

Guzel-Seydim, Z. B., Gökırmaklı, Ç., & Greene, A. K. (2021). A comparison of milk

kefir and water kefir: Physical, chemical, microbiological and functional
properties. Trends in Food Science & Technology, 113, 42–53.
https://doi.org/10.1016/j.tifs.2021.04.041

Himmetagaoglu, A. B., & Erbay, Z. (2019). Effects of spray drying process conditions
on the quality properties of microencapsulated cream powder. International Dairy
Journal, 88, 60–70. https://doi.org/10.1016/j.idairyj.2018.08.004

Ilıkkan, Ö. K., & Bağdat, E. Ş. (2021). Comparison of bacterial and fungal biodiversity
of Turkish kefir grains with high-throughput metagenomic analysis. LWT, 152,
112375. https://doi.org/10.1016/j.lwt.2021.112375

Instituto Adolfo Lutz. (2008). Métodos Físico-Químicos para Análise de Alimentos (4a
(1a ed. digital)).
 39

http://www.ial.sp.gov.br/resources/editorinplace/ial/2016_3_19/analisedealimentos
ial_2008.pdf

Jinapong, N., Suphantharika, M., & Jamnong, P. (2008). Production of instant soymilk
powders by ultrafiltration, spray drying and fluidized bed agglomeration. Journal
of Food Engineering, 84(2), 194–205.
https://doi.org/10.1016/j.jfoodeng.2007.04.032

Kalamaki, M. S., & Angelidis, A. S. (2017). Isolation and molecular identification of

yeasts in Greek kefir. International Journal of Dairy Technology, 70(2), 261–268.
https://doi.org/10.1111/1471-0307.12329

Kandylis, P., Pissaridi, K., Bekatorou, A., Kanellaki, M., & Koutinas, A. A. (2016).
Dairy and non-dairy probiotic beverages. Current Opinion in Food Science, 7, 58–
63. https://doi.org/10.1016/j.cofs.2015.11.012

Kuck, L. S., & Noreña, C. P. Z. (2016). Microencapsulation of grape (Vitis labrusca

var. Bordo) skin phenolic extract using gum Arabic, polydextrose, and partially
hydrolyzed guar gum as encapsulating agents. Food Chemistry, 194, 569–576.
https://doi.org/10.1016/j.foodchem.2015.08.066

Laureys, D., Aerts, M., Vandamme, P., & De Vuyst, L. (2018). Oxygen and diverse
nutrients influence the water kefir fermentation process. Food Microbiology, 73,
351–361. https://doi.org/10.1016/j.fm.2018.02.007

Laureys, D., Aerts, M., Vandamme, P., & De Vuyst, L. (2019). The Buffer Capacity
and Calcium Concentration of Water Influence the Microbial Species Diversity,
Grain Growth, and Metabolite Production During Water Kefir Fermentation.
Frontiers in Microbiology, 10. https://doi.org/10.3389/fmicb.2019.02876

Laureys, D., & De Vuyst, L. (2014). Microbial Species Diversity, Community

Dynamics, and Metabolite Kinetics of Water Kefir Fermentation. Applied and
Environmental Microbiology, 80(8), 2564–2572.
https://doi.org/10.1128/AEM.03978-13

Laureys, D., & De Vuyst, L. (2017). The water kefir grain inoculum determines the
characteristics of the resulting water kefir fermentation process. Journal of Applied
Microbiology, 122(3), 719–732. https://doi.org/10.1111/jam.13370

Laureys, D., Van Jean, A., Dumont, J., & De Vuyst, L. (2017). Investigation of the
instability and low water kefir grain growth during an industrial water kefir
fermentation process. Applied Microbiology and Biotechnology, 101(7), 2811–
2819. https://doi.org/10.1007/s00253-016-8084-5

Lombard, J. (2016). Characterisation of Wickerhamomyces anomalus and Kazachstania

aerobia: Investigating fermentation kinetics and aroma production [Master of
Science]. Stellenbosch University.

Ma, D., Wang, B., Xiao, S., & Wang, J. (2024). Non-targeted metabolomics unveils
metabolic spectrum characteristics and core metabolite interaction networks in
 40

water kefir fermentation. LWT, 195, 115840.

https://doi.org/10.1016/j.lwt.2024.115840

Magalhães, K. T., de M. Pereira, G. V., Dias, D. R., & Schwan, R. F. (2010). Microbial
communities and chemical changes during fermentation of sugary Brazilian kefir.
World Journal of Microbiology and Biotechnology, 26(7), 1241–1250.
https://doi.org/10.1007/s11274-009-0294-x

Maia, J. L. (2019). Microencapsulação do extrato do jambo vermelho (Syzygium

malaccense L.) utilizando spray dryer e liofilizador [Tese de Doutorado].
Universidade Federal do Rio Grande do Norte.

Marsh, A. J., Hill, C., Ross, R. P., & Cotter, P. D. (2014). Fermented beverages with
health-promoting potential: Past and future perspectives. Trends in Food Science &
Technology, 38(2), 113–124. https://doi.org/10.1016/j.tifs.2014.05.002

Martínez-Cervera, S., Salvador, A., Muguerza, B., Moulay, L., & Fiszman, S. M.
(2011). Cocoa fibre and its application as a fat replacer in chocolate muffins. LWT
- Food Science and Technology, 44(3), 729–736.
https://doi.org/10.1016/j.lwt.2010.06.035

Montanuci, F. D. (2010). Bebidas de Kefir com e sem inulina em versões integral e

desnatada: elaboração e caracterização química, física, microbiológica e
sensorial [Programa de Mestrado em Ciência de Alimentos]. Universidade
Estadual de Londrina.

Nale, Z., Tontul, I., Aşçi Arslan, A., Sahin Nadeem, H., & Kucukcetin, A. (2018).
Microbial viability, physicochemical and sensory properties of kefir microcapsules
prepared using maltodextrin/Arabic gum mixes. International Journal of Dairy
Technology, 71, 61–72. https://doi.org/10.1111/1471-0307.12402

Olgenblum, G. I., Sapir, L., & Harries, D. (2020). Properties of Aqueous Trehalose
Mixtures: Glass Transition and Hydrogen Bonding. Journal of Chemical Theory
and Computation, 16(2), 1249-1262. https://doi.org/10.1021/acs.jctc.9b01071

Ouyang, W., Liao, Z., Yang, X., Zhang, X., Zhu, X., Zhong, Q., Wang, L., Fang, X., &
Wang, J. (2024). Microbial Composition of Water Kefir Grains and Their
Application for the Detoxification of Aflatoxin B1. Toxins, 16(2), 107.
https://doi.org/10.3390/toxins16020107

Paiva, D. L., Lampman, G. M., Kriz, G. S., & Vyvyan, J. R. (2013). Introdução à
espectroscopia (5a Edição). Cengage Learning.

Patel, S. H., Tan, J. P., Börner, R. A., Zhang, S. J., Priour, S., Lima, A., Ngom-Bru, C.,
Cotter, P. D., & Duboux, S. (2022). A temporal view of the water kefir microbiota
and flavour attributes. Innovative Food Science & Emerging Technologies, 80,
103084. https://doi.org/10.1016/j.ifset.2022.103084
 41

Porter, T. J., Divol, B., & Setati, M. E. (2019). Lachancea yeast species: Origin,
biochemical characteristics and oenological significance. Food Research
International, 119, 378–389. https://doi.org/10.1016/j.foodres.2019.02.003

Prado, F. C., Parada, J. L., Pandey, A., & Soccol, C. R. (2008). Trends in non-dairy
probiotic beverages. Food Research International, 41(2), 111–123.
https://doi.org/10.1016/j.foodres.2007.10.010

Preethi, R., Moses, J. A., & Anandharamakrishnan, C. (2021). Effect of conductive

hydro-drying on physiochemical and functional properties of two pulse protein
extracts: Green gram (Vigna radiata) and black gram (Vigna mungo). Food
Chemistry, 343, 128551. https://doi.org/10.1016/j.foodchem.2020.128551

Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., &
Glöckner, F. O. (2012). The SILVA ribosomal RNA gene database project:
improved data processing and web-based tools. Nucleic Acids Research, 41(D1),
D590–D596. https://doi.org/10.1093/nar/gks1219

Randazzo, W., Corona, O., Guarcello, R., Francesca, N., Germanà, M. A., Erten, H.,
Moschetti, G., & Settanni, L. (2016). Development of new non-dairy beverages
from Mediterranean fruit juices fermented with water kefir microorganisms. Food
Microbiology, 54, 40–51. https://doi.org/10.1016/j.fm.2015.10.018

Rodríguez, M. A., Fernández, L. A., Díaz, M. L., Pérez, M., Corona, M., & Reynaldi, F.
J. (2023). Microbiological and chemical characterization of water kefir: An
innovative source of potential probiotics for bee nutrition. Revista Argentina de
Microbiología, 55(2), 176–180. https://doi.org/10.1016/j.ram.2022.09.003

Romano, N., Mobili, P., Zuñiga-Hansen, M. E., & Gómez-Zavaglia, A. (2018).

Physico-chemical and structural properties of crystalline inulin explain the stability
of Lactobacillus plantarum during spray-drying and storage. Food Research
International, 113, 167–174. https://doi.org/10.1016/j.foodres.2018.07.007

Sakoui, S., Derdak, R., Pop, O. L., Vodnar, D. C., Addoum, B., Teleky, B.-E., Elemer,
S., Elmakssoudi, A., Suharoschi, R., Soukri, A., & El Khalfi, B. (2022). Effect of
encapsulated probiotic in Inulin-Maltodextrin-Sodium alginate matrix on the
viability of Enterococcus mundtii SRBG1 and the rheological parameters of
fermented milk. Current Research in Food Science, 5, 1713–1719.
https://doi.org/10.1016/j.crfs.2022.09.027

Sanches, F. L., Weis, C. M. S. C., Gonçalves, G. C. V., Andrade, G. S., Diniz, L. G. T.,
Camargo, A. F., Kubeneck, S., Klein, G. H., Romani, L. C., Longo, V. D., Bürck,
M., Tormen, L., Braga, A. R. C., Francisco, C. T. D. P., Treichel, H., & Bertan, L.
C. (2024). Study and characterization of a product based on a vegetable extract of
quinoa fermented with water kefir grains. World Journal of Microbiology and
Biotechnology, 40(4), 118. https://doi.org/10.1007/s11274-024-03943-x

Santivarangkna, C., Kulozik, U., & Foerst, P. (2008). Inactivation mechanisms of lactic
acid starter cultures preserved by drying processes. Journal of Applied
Microbiology, 105(1), 1–13. https://doi.org/10.1111/j.1365-2672.2008.03744.x
 42

Schmidt, P.-A., Bálint, M., Greshake, B., Bandow, C., Römbke, J., & Schmitt, I. (2013).
Illumina metabarcoding of a soil fungal community. Soil Biology and
Biochemistry, 65, 128–132. https://doi.org/10.1016/j.soilbio.2013.05.014

Silva, C. R. P. (2021). Secagem por atomização do extrato de sapotado-solimões

(Matisia cordata) [Dissertação - Mestrado em Ciência dos Alimentos].
Universidade Federal de Lavras.

Silva, C. F. G. Da, Santos, F. L., Santana, L. R. R. De, Silva, M. V. L., & Conceição, T.
De A. (2018). Development and characterization of a soymilk Kefir-based
functional beverage. Food Science and Technology, 38(3), 543–550.
https://doi.org/10.1590/1678-457x.10617

Tabatabaei, M., Ebrahimi, B., Rajaei, A., Movahednejad, M. H., Rastegari, H., Taghavi,
E., Aghbashlo, M., Gupta, V. K., & Lam, S. S. (2022). Producing submicron
chitosan-stabilized oil Pickering emulsion powder by an electrostatic collector-
equipped spray dryer. Carbohydrate Polymers, 294, 119791.
https://doi.org/10.1016/j.carbpol.2022.119791

Tapia, M. S., Alzamora, S. M., & Chirife, J. (2020). Effects of water activity (𝑎𝑤) on
microbial stability as a hurdle in food preservation. In G. V. Barbosa-Cánovas, A.
J. Fontana Jr., S. J. Schmidt, & T. P. Labuza (Eds.), Water activity in foods:
Fundamentals and applications (2nd ed., Chapter 14).
https://doi.org/10.1002/9781118765982.ch14

Teijeiro, M., Pérez, P. F., De Antoni, G. L., & Golowczyc, M. A. (2018). Suitability of
kefir powder production using spray drying. Food Research International, 112,
169–174. https://doi.org/10.1016/j.foodres.2018.06.023

Teunou, E., Fitzpatrick, J. J., & Synnott, E. C. (1999). Characterisation of food powder
flowability. Journal of Food Engineering, 39(1), 31–37.
https://doi.org/10.1016/S0260-8774(98)00140-X

Thamacharoensuk, T., Kitahara, M., Ohkuma, M., Thongchul, N., & Tanasupawat, S.
(2015). Sporolactobacillus shoreae sp. nov. and Sporolactobacillus spathodeae sp.
nov., two spore-forming lactic acid bacteria isolated from tree barks in Thailand.
International Journal of Systematic and Evolutionary Microbiology, 65(Pt_4),
1220–1226. https://doi.org/10.1099/ijs.0.000084

Tireki, S. (2024). Influence of Water Properties on the Physicochemical and Sensorial

Parameters of Water Kefir. Journal of Culinary Science & Technology, 1–21.
https://doi.org/10.1080/15428052.2024.2303002

Tontul, I., Ergin, F., Eroğlu, E., Küçükçetin, A., & Topuz, A. (2021). The impact of
refractance window drying conditions on the physical and microbiological
properties of kefir powder. Food Bioscience, 43, 101317.
https://doi.org/10.1016/j.fbio.2021.101317
 43

Tontul, I., & Topuz, A. (2017). Spray-drying of fruit and vegetable juices: Effect of
drying conditions on the product yield and physical properties. Trends in Food
Science & Technology, 63, 91–102. https://doi.org/10.1016/j.tifs.2017.03.009

Verruck, S., de Liz, G. R., Dias, C. O., Amboni, R. D. M. C., & Prudencio, E. S. (2019).
Effect of full-fat goat's milk and prebiotics use on Bifidobacterium BB-12 survival
and on the physical properties of spray-dried powders under storage conditions.
Food Research International, 119, 643-652.
https://doi.org/10.1016/j.foodres.2018.10.042

Verruck, S., Prudêncio, E. S., Müller, C. M. O., Fritzen-Freire, C. B., & Amboni, R. D.
de M. C. (2015). Influence of Bifidobacterium Bb-12 on the physicochemical and
rheological properties of buffalo Minas Frescal cheese during cold storage. Journal
of Food Engineering, 151, 34–42. https://doi.org/10.1016/j.jfoodeng.2014.11.021

Verruck, S., Santana, F., de Olivera Müller, C., & Prudencio, E. S. (2018). Thermal and
water sorption properties of Bifidobacterium BB-12 microcapsules obtained from
goat's milk and prebiotics. LWT, 98, 314–321.
https://doi.org/10.1016/j.lwt.2018.08.060

Villanoeva, C. N. B., DL, R., RL, A., LB, A., SHC, S., AC, N., JR, N., & E, N. (2021).
Functionally Active Microbiome and Physicochemical Properties of Milk and
Sugary Water Kefir from Brazil. Austin Food Sciences, 6(1).
https://doi.org/10.26420/austinfoodsci.2021.1042

Waldherr, F. W., Doll, V. M., Meißner, D., & Vogel, R. F. (2010). Identification and
characterization of a glucan-producing enzyme from Lactobacillus hilgardii TMW
1.828 involved in granule formation of water kefir. Food Microbiology, 27(5),
672–678. https://doi.org/10.1016/j.fm.2010.03.013

Wang, Y., & Qian, P.-Y. (2009). Conservative Fragments in Bacterial 16S rRNA Genes
and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies.
PLoS ONE, 4(10), e7401. https://doi.org/10.1371/journal.pone.0007401

Wang, Y., Truong, T., Li, H., & Bhandari, B. (2019). Co-melting behaviour of sucrose,
glucose &amp; fructose. Food Chemistry, 275, 292–298.
https://doi.org/10.1016/j.foodchem.2018.09.109

Weir, P. M. (2016). The ecology of Zymomonas: a review. Folia Microbiologica, 61(5),

385–392. https://doi.org/10.1007/s12223-016-0447-x

White, T. J., Bruns, S. B., Lee, S. B., & Taylor, J. W. (1990). Amplification and Direct
Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In PCR
Protocols, a Guide to Methods and Applications (pp. 315–322). Academic Press.

Xu, D., Bechtner, J., Behr, J., Eisenbach, L., Geißler, A. J., & Vogel, R. F. (2019).
Lifestyle of Lactobacillus hordei isolated from water kefir based on genomic,
proteomic and physiological characterization. International Journal of Food
Microbiology, 290, 141–149. https://doi.org/10.1016/j.ijfoodmicro.2018.10.004
 44

Yerlikaya, O., Akan, E., & Kinik, Ö. (2022). The metagenomic composition of water
kefir microbiota. International Journal of Gastronomy and Food Science, 30,
100621. https://doi.org/10.1016/j.ijgfs.2022.100621

Yıldız, S., Reisoglu, Ş., & Aydin, S. (2024). Evaluation of functional quality of water
kefir with Haematococcus pluvialis biomass and colour pigment astaxanthin.
International Journal of Food Science & Technology, 59(6), 4326–4335.
https://doi.org/10.1111/ijfs.16975

Zongo, O., Cruvellier, N., Leray, F., Bideaux, C., Lesage, J., Zongo, C., Traoré, Y.,
Savadogo, A., & Guillouet, S. (2020). Physicochemical composition and
fermentation kinetics of a novel Palm Sap-based Kefir Beverage from the
fermentation of Borassus aethiopum Mart. fresh sap with kefir grains and ferments.
Scientific African, 10, e00631. https://doi.org/10.1016/j.sciaf.2020.e00631

Declaration of interests

☒ The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.

☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
 45

Graphical abstract