Kefir Probiotic-Enriched Non-Alcoholic Beers: Microbial, Genetic, and Sensory-Chemical Assessment
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Substrate for Sugary Kefir Grains Adaptation
2.3. Inoculation Process of Sugary Kefir Grains in Commercial Non-Alcoholic Beers
2.4. Fermentation Kinetics: Physicochemical Analyses
2.5. Microbial Genetic Profile Analysis and Species-Level Detection
2.6. Comparative Analysis of Color Scales for Non-Alcoholic Beers According to the EBC (European Brewing Convention) and SRM (Standard Reference Method)
2.7. Sensory Analysis
2.8. Microbial Shelf Life Analysis of Fermented Non-Alcoholic Beers
2.9. Statistics
3. Results
3.1. Sugary Kefir Grains Adaptation
3.2. Fermentation Kinetics
3.2.1. Sugar and Acidity Parameters
3.2.2. Organic Acids and Ethanol Parameters
3.2.3. Microbial Genetic Profile
3.2.4. Color Scale for Non-Alcoholic Beers
3.2.5. Sensory Parameters
3.3. Microbial Shelf Life for Non-Alcoholic Beers Fermented with Sugary Kefir
4. Discussion
5. Conclusions
6. Limitations of the Study and Future Prospects
- (1)
- A comparative analysis using multiple commercial non-alcoholic beer brands to evaluate matrix variability;
- (2)
- Longitudinal studies assessing probiotic viability, physicochemical stability, and sensory quality over shelf life;
- (3)
- In-depth characterization of sensory profiles using trained and consumer panels;
- (4)
- Exploration of encapsulation or stabilization techniques to enhance the survival of probiotics in beer matrices.
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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pH | Acidity (% m/v) | |||
---|---|---|---|---|
0 h | 24 h | 0 h | 24 h | |
Control NAB1 | 3.72 aA ± 0.03 | 3.72 aA ± 0.01 | 1.29 cC ± 0.02 | 1.22 cC ± 0.02 |
NAB1 with kefir inoculant | 3.71 aA ± 0.02 | 3.52 bB ± 0.01 | 1.31 cC ± 0.02 | 2.35 dD ± 0.02 |
Control NAB2 | 3.74 aA ± 0.01 | 3.73 aA ± 0.01 | 1.27 cC ± 0.02 | 1.26 cC ± 0.02 |
NAB2 with kefir inoculant | 3.71 aA ± 0.02 | 3.55 bB ± 0.01 | 1.30 cC ± 0.02 | 2.33 dD ± 0.01 |
Control NAB3 | 3.75 aA ± 0.01 | 3.75 aA ± 0.01 | 1.28 cC ± 0.02 | 1.25 cC ± 0.02 |
NAB3 with kefir inoculant | 3.74 aA ± 0.01 | 3.59 bB ± 0.01 | 1.31 cC ± 0.02 | 2.35 dC ± 0.02 |
°Brix | Sucrose (g/L) | |||
0 h | 24 h | 0 h | 24 h | |
Control NAB1 | 5.57 eE ± 0.01 | 5.47 eE ± 0.02 | 0.34 gG ± 0.01 | 0.34 gG ± 0.01 |
NAB1 with kefir inoculant | 5.57 eE ± 0.01 | 3.80 fF ± 0.02 | 0.35 gG ± 0.01 | 0.22 hH ± 0.01 |
Control NAB2 | 5.55 eE ± 0.01 | 5.51 eE ± 0.02 | 0.35 gG ± 0.01 | 0.34 gG ± 0.01 |
NAB2 with kefir inoculant | 5.55 eE ± 0.01 | 3.78 fF ± 0.02 | 0.34 gG ± 0.01 | 0.22 hH ± 0.01 |
Control NAB3 | 5.55 eE ± 0.01 | 5.50 eE ± 0.02 | 0.36 gA ± 0.01 | 0.35 gG ± 0.01 |
NAB3 with kefir inoculant | 5.55 eE ± 0.01 | 3.77 fF ± 0.02 | 0.36 gA ± 0.01 | 0.23 hH ± 0.01 |
Samples | Lactic Acid (g/L) | Acetic Acid (g/L) | Ethanol (g/L) | |
---|---|---|---|---|
0 h | Control NAB1 | 0.54 ± 0.01 aA | 0.09 ± 0.01 bB | ND cC |
NAB1 with kefir inoculant | 0.55 ± 0.01 aA | 0.07 ± 0.01 bB | ND cC | |
Control NAB2 | 0.56 ± 0.01 aA | 0.09 ± 0.01 bB | ND cC | |
NAB2 with kefir inoculant | 0.55 ± 0.01 aA | 0.09 ± 0.01 bB | ND cC | |
Control NAB3 | 0.56 ± 0.01 aA | 0.08 ± 0.01 bB | ND cC | |
NAB3 with kefir inoculant | 0.54 ± 0.01 aA | 0.08 ± 0.01 bB | ND cC | |
24 h | Control NAB1 | 0.54 ± 0.01 aA | 0.09 ± 0.01 bB | ND cC |
NAB1 with kefir inoculant | 1.25 ± 0.02 dD | 0.12 ± 0.01 eE | 0.02 ± 0.01 fF | |
Control NAB2 | 0.54 ± 0.01 aA | 0.08 ± 0.01 bB | ND cC | |
NAB2 with kefir inoculant | 1.28 ± 0.06 dD | 0.14 ± 0.01 eE | 0.03 ± 0.00 fF | |
Control NAB3 | 0.54 ± 0.01 aA | 0.09 ± 0.01 bB | ND cC | |
NAB3 with kefir inoculant | 1.30 ± 0.02 dD | 0.14 ± 0.01 eE | 0.02 ± 0.01 fF |
Band | Prokaryotic Species | NCBI-BLAST | Similarity (%) | e-Value |
---|---|---|---|---|
A | Lacticaseibacillus paracasei | AB368902.1 | 99 | <10e-100 |
B | Lacticaseibacillus casei | EU626005.1 | 98 | <10e-50 |
C | Lacticaseibacillus paracasei subsp. paracasei | NR025880.1 | 98 | <10e-50 |
D | Lacticaseibacillus paracasei subsp. tolerans | AB181950.1 | 99 | <10e-100 |
E | Lactobacillus delbrueckii subsp. lactis | EU194346.1 | 98 | <10e-50 |
F | Lentilactobacillus parabuchneri | AB368914.1 | 99 | <10e-100 |
G | Lentilactobacillus kefiri | AB3626680.1 | 99 | <10e-100 |
H | Lactococcus lactis | EU194346.1 | 99 | <10e-100 |
I | Leuconostoc citreum | FJ378896.1 | 99 | <10e-100 |
J | Acetobacter lovaniensis | AB308060.1 | 99 | <10e-100 |
Band | Eukaryotic Species | NCBI-BLAST | Similarity (%) | e-value |
K | Saccharomyces cerevisiae | EU649673.1 | 99 | <10e-100 |
L | Kluyveromyces lactis | AJ229069.1 | 99 | <10e-100 |
M | Lachancea meyersii | AY645661.1 | 99 | <10e-100 |
N | Kazachstania aerobia | AY582126.1 | 99 | <10e-100 |
Microbiology | T0—Fermented Non-Alcoholic Beers | T10 Days | T20 Days | T30 Days |
---|---|---|---|---|
NAB1 | 4 °C /mL | 4 °C /mL | 4 °C /mL | 4 °C /mL |
Lactobacillus, Lacticaseibacillus and Lentilactobacillus | # 1.8 × 107 CFU ± 0.02 | # 1.8 × 107 CFU ± 0.02 | # 1.2 × 107 CFU ± 0.02 | # 1.1 × 107 CFU ± 0.02 |
Lactococcus | 1.4 × 105 CFU ± 0.02 | 1.4 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 |
Leuconostoc | 1.4 × 106 CFU ± 0.02 | 1.4 × 106 CFU ± 0.02 | 1.4 × 106 CFU ± 0.02 | 1.4 × 105 CFU ± 0.02 |
Streptococcus | ND | ND | ND | ND |
Acetobacter | 1.2 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 104 CFU ± 0.02 |
Yeasts | # 5.3 × 108 CFU ± 0.02 | # 5.1 × 108 CFU ± 0.02 | # 5.1 × 108 CFU ± 0.02 | # 4.3 × 107 CFU ± 0.02 |
NAB1 | 25 °C /mL | 25 °C /mL | 25 °C /mL | 25 °C /mL |
Lactobacillus, Lacticaseibacillus and Lentilactobacillus | # 1.8 × 107 CFU ± 0.02 | # 1.2 × 107 CFU ± 0.02 | # 1.1 × 107 CFU ± 0.02 | # 1.1 × 107 CFU ± 0.02 |
Lactococcus | 1.4 × 105 CFU ± 0.02 | 1.2 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 |
Leuconostoc | 1.4 × 106 CFU ± 0.02 | 1.3 × 106 CFU ± 0.02 | 1.2 × 106 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 |
Streptococcus | ND | ND | ND | ND |
Acetobacter | 1.2 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 104 CFU ± 0.02 | 1.1 × 104 CFU ± 0.02 |
Yeasts | # 5.3 × 108 CFU ± 0.02 | # 5.1 × 108 CFU ± 0.02 | # 5.1 × 107 CFU ± 0.02 | # 4.3 × 107 CFU ± 0.02 |
NAB2 | 4 °C /mL | 4 °C /mL | 4 °C /mL | 4 °C /mL |
Lactobacillus, Lacticaseibacillus and Lentilactobacillus | # 1.8 × 107 CFU ± 0.02 | # 1.8 × 107 CFU ± 0.02 | # 1.2 × 107 CFU ± 0.02 | # 1.1 × 107 CFU ± 0.02 |
Lactococcus | 1.4 × 105 CFU ± 0.02 | 1.4 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 |
Leuconostoc | 1.4 × 106 CFU ± 0.02 | 1.4 × 106 CFU ± 0.02 | 1.4 × 106 CFU ± 0.02 | 1.4 × 105 CFU ± 0.02 |
Streptococcus | ND | ND | ND | ND |
Acetobacter | 1.2 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 104 CFU ± 0.02 | 1.2 × 104 CFU ± 0.02 |
Yeasts | # 5.3 × 108 CFU ± 0.02 | # 5.1 × 108 CFU ± 0.02 | # 5.2 × 108 CFU ± 0.02 | # 4.1 × 107 CFU ± 0.02 |
NAB2 | 25 °C /mL | 25 °C /mL | 25 °C /mL | 25 °C /mL |
Lactobacillus, Lacticaseibacillus and Lentilactobacillus | # 1.8 × 107 CFU ± 0.02 | # 1.2 × 107 CFU ± 0.02 | # 1.1 × 107 CFU ± 0.02 | # 1.1 × 107 CFU ± 0.02 |
Lactococcus | 1.4 × 105 CFU ± 0.02 | 1.2 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 |
Leuconostoc | 1.4 × 106 CFU ± 0.02 | 1.3 × 106 CFU ± 0.02 | 1.2 × 106 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 |
Streptococcus | ND | ND | ND | ND |
Acetobacter | 1.2 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 104 CFU ± 0.02 | 1.1 × 104 CFU ± 0.02 |
Yeasts | # 5.3 × 108 CFU ± 0.02 | # 5.1 × 108 CFU ± 0.02 | # 4.1 × 107 CFU ± 0.02 | # 3.3 × 107 CFU ± 0.02 |
NAB3 | 4 °C /mL | 4 °C /mL | 4 °C /mL | 4 °C /mL |
Lactobacillus, Lacticaseibacillus and Lentilactobacillus | # 1.8 × 107 CFU ± 0.02 | # 1.8 × 107 CFU ± 0.02 | # 1.2 × 107 CFU ± 0.02 | # 1.1 × 107 CFU ± 0.02 |
Lactococcus | 1.4 × 105 CFU ± 0.02 | 1.4 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 |
Leuconostoc | 1.4 × 106 CFU ± 0.02 | 1.4 × 106 CFU ± 0.02 | 1.4 × 106 CFU ± 0.02 | 1.4 × 105 CFU ± 0.02 |
Streptococcus | ND | ND | ND | ND |
Acetobacter | 1.2 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 104 CFU ± 0.02 | 1.2 × 104 CFU ± 0.02 |
Yeasts | # 5.3 × 108 CFU ± 0.02 | # 5.1 × 108 CFU ± 0.02 | # 5.1 × 107 CFU ± 0.02 | # 4.4 × 107 CFU ± 0.02 |
NAB3 | 25 °C /mL | 25 °C /mL | 25 °C /mL | 25 °C /mL |
Lactobacillus, Lacticaseibacillus and Lentilactobacillus | # 1.8 × 107 CFU ± 0.02 | # 1.2 × 107 CFU ± 0.02 | # 1.1 × 107 CFU ± 0.02 | # 1.1 × 107 CFU ± 0.02 |
Lactococcus | 1.4 × 105 CFU ± 0.02 | 1.2 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 |
Leuconostoc | 1.4 × 106 CFU ± 0.02 | 1.3 × 106 CFU ± 0.02 | 1.2 × 106 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 |
Streptococcus | ND | ND | ND | ND |
Acetobacter | 1.2 × 105 CFU ± 0.02 | 1.1 × 105 CFU ± 0.02 | 1.1 × 104 CFU ± 0.02 | 1.1 × 104 CFU ± 0.02 |
Yeasts | # 5.3 × 108 CFU ± 0.02 | # 5.1 × 108 CFU ± 0.02 | # 4.2 × 107 CFU ± 0.02 | # 3.2 × 107 CFU ± 0.02 |
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do Nascimento, A.S.M.; da Silva, R.N.A.; Tavares, P.P.L.G.; Borges, A.S.; Cardoso, M.P.S.; Lobato, A.K.d.C.L.; Almeida, R.C.d.C.; Magalhães-Guedes, K.T. Kefir Probiotic-Enriched Non-Alcoholic Beers: Microbial, Genetic, and Sensory-Chemical Assessment. Beverages 2025, 11, 75. https://doi.org/10.3390/beverages11030075
do Nascimento ASM, da Silva RNA, Tavares PPLG, Borges AS, Cardoso MPS, Lobato AKdCL, Almeida RCdC, Magalhães-Guedes KT. Kefir Probiotic-Enriched Non-Alcoholic Beers: Microbial, Genetic, and Sensory-Chemical Assessment. Beverages. 2025; 11(3):75. https://doi.org/10.3390/beverages11030075
Chicago/Turabian Styledo Nascimento, Alessandra Souza Marques, Raquel Nunes Almeida da Silva, Pedro Paulo Lordelo Guimarães Tavares, Adriana Silva Borges, Marina Passos Soares Cardoso, Ana Katerine de Carvalho Lima Lobato, Rogéria Comastri de Castro Almeida, and Karina Teixeira Magalhães-Guedes. 2025. "Kefir Probiotic-Enriched Non-Alcoholic Beers: Microbial, Genetic, and Sensory-Chemical Assessment" Beverages 11, no. 3: 75. https://doi.org/10.3390/beverages11030075
APA Styledo Nascimento, A. S. M., da Silva, R. N. A., Tavares, P. P. L. G., Borges, A. S., Cardoso, M. P. S., Lobato, A. K. d. C. L., Almeida, R. C. d. C., & Magalhães-Guedes, K. T. (2025). Kefir Probiotic-Enriched Non-Alcoholic Beers: Microbial, Genetic, and Sensory-Chemical Assessment. Beverages, 11(3), 75. https://doi.org/10.3390/beverages11030075