Kombucha: Production and Microbiological Research †
Abstract
:1. Introduction
2. Microbiological Characteristics of Kombucha
2.1. Presence of Acetic Acid Bacteria in Fermented Foods
2.1.1. AAB Used in the Production of Fermented Products
2.1.2. Dominant Acetic Acid Bacteria Found in Kombucha
2.2. Lactic Acid Bacteria Isolated from Kombucha
2.3. Yeast Isolated from Kombucha
2.3.1. General Characteristics of Yeast
2.3.2. Dominant Yeast Present in Kombucha
3. Isolation of AAB and Yeast from Kombucha
3.1. Isolation, Enumeration and Preservation of AAB
3.2. Isolation and Enumeration of Yeast
4. Phenotypic Characterisation and Identification of Acetic Acid Bacteria and Yeast from Kombucha
4.1. Phenotypic Characterisation of AAB
4.2. Phenotypic Characterisation and Identification of Yeast from Kombucha
Identification of Yeast Using Commercial Kits
5. Genotypic Identification of AAB and Yeast from Kombucha
5.1. Genotypic Identification of AAB from Kombucha
5.2. Genotypic Identification of Yeast
6. Conclusions and Future Research on Kombucha
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Brand | Origin | Packaging | Flavour(s) | Storage Conditions |
---|---|---|---|---|
Remedy | Australia | 330 mL (glass bottles) 250 mL (cans) 1.25 L (plastic bottles) | Ginger lemon, Raspberry Lemonade, Cola, Wildberry, Mango Passion, Passionfruit, Apple Crisp, Peach | Chilled/ambient temperature |
Batchwell | New Zealand | 375 mL (glass bottles) | Braeburn apple, Pineapple and Ginger, Motueka hops, Ginger and Turmeric, Early grey | Chilled |
Daily Organics | New Zealand | 200 mL (glass bottles) 1 L (glass bottles) | Original Kombucha, Chai spices and ginger, Lemon and ginger | Chilled |
LO BROS | New Zealand | 330 mL (glass bottles) 250 mL (cans) 750 mL (glass bottles) | Feijoa, Raspberry and lemon, Orange and mango, Ginger and lemon, Mango, Ginger and turmeric, Blueberry, Passionfruit, Cola, Ginger beer, Lemon lime and bitters, Pineapple and lime | Chilled/ ambient temperature |
MAMA’S Brew Shop | New Zealand | 375 mL (glass bottles) 330 mL (cans) | Lemongrass and ginger, Lavender and hibiscus | Chilled |
Karma Drinks | New Zealand | 330 mL (glass bottles) | Lemon and ginger, Raspberry and lemon, Mango and passionfruit, Cherryand berry | Chilled |
Good Buzz | New Zealand | 250 mL (cans) 328 mL (glass bottles) 375 mL (glass bottles) 888 mL (glass bottles) | Passionfruit and guava, Blueberry and peach, Pineapple and mango, Feijoa, Raspberry and lemon, Mango, Gisborne lemon and Manuka leaf, Hawkes Bay, peach and kawakawa | Chilled or ambient temperature |
Species | Region | Reference |
---|---|---|
Lacticaseibacillus casei | NS | [35] |
Lactiplantibacillus plantarum | China | [38] |
Lactobacillus nagelii | United State of America (USA) | [39] |
Lactobacillus rhamnosus | NS | [5] |
Lactobacillus mali | USA | [39] |
Pediococcus (P.) pentosaceus | Romania | [40] |
P.acidiliactici | Romania | [36] |
Species | Morphology | Characteristics |
---|---|---|
Zygosaccharomyces (Z.) bailii [1] | White to cream colonies with brownish top, cylindrical or ellipsoidal shape, (3.5–6.0) × (4.5–11.5) μm in size | Tolerant to organic acids, Forms acetic acid, heat tolerance < 75 °C Growth pH > 2 and < 7 [48] |
Zycosaccharomyces (Z.) rouxii | White to cream smooth colonies, round or oval shape | High osmotic stress and salt/sugar tolerant, grows under low oxygen and low water activity [49] |
Schizosaccharomyces (S.) pombe | Cream to tan, butyrous colonies, rod-shaped | Can convert malic acid to ethanol, high resistance to low water activity, low pH and wide range of temperature environments, highly sugar content tolerant [50] |
Saccharomycodes (S.) ludwigi [10] | Cream, butyrous colonies, elongated shape, and swelling in the centre | Resistant to pressurised carbon dioxide, high sugar tolerant [51] |
S. cerevisiae [27] | White to cream, butyrous colonies, spherical or ovoid shape, 2.5–10.0 µm (diameter) | Can convert glucose to ethanol, high ethanol tolerance, rapid fermentation rate [52] |
Brettanomyces (B.) bruxellensis [24,47] | Distinctive elongated shape, 2.5–10.0 µm (diameter) | Can produce high amounts of acetic acid and ethanol under aerobic conditions, high ethanol concentration (up to 15%), able to grow under low pH and oxygen environment, high efficiency to utilise nitrogen sources [53] |
Species | Country | References |
---|---|---|
Brettanomyces (B.) anamalus | New Zealand | [28] |
B. lambicus | Germany | [54] |
B. custerisii | Germany | [54] |
B. intermedius | NS | [24] |
B. claussenii | NS | [24] |
Candida (C.) albican C. colleculosa C. kefir C. krusei | Japan Saudi Arabia Saudi Arabia Saudi Arabia | [24] [55] [55] [55] |
C. guilliermondii C. obtuse C. stellata | Japan/Saudi Arabia Formosa Australia | [56] [46] [46] |
C. famata | Indonesia | [57] |
C. magnoliae | Indonesia | [57] |
Dekkera (D.) bruxelensis | New Zealand | [28] |
Hanseniaspora (H.) valbyensis | New Zealand | [28] |
Lachancea (L.) fermentati | USA | [58] |
Kloeckera (K.) apiculata Kluyceromyces (K.) africanus | NS NS | [24] [24] |
Pichia (P.) fermentans | NS | [4] |
P. membranefaciens | NS | [26] |
P. kudriavzevii | New Zealand | [28] |
Torulaspora (T.) delbrueckii | Australia | [46] |
Torulopsis (T.)famata | Japan | [56] |
Zygosaccharomyces (Z.) kombucahensis | Russia | [59] |
Characteristics | Acetobacter | Gluconacetobacter | Gluconobacter | Komagataeibacter |
---|---|---|---|---|
Cell shape | Ellipsoidal to rods | Ellipsoidal to rods | Ellipsoidal to Rods | Coccoid to rods |
Cell size (μm) | 0.4–1.0 × 1.0–3.0 | 0.5–0.9 × 1.0–2.0 | 0.6–1.0 × 1.0–3.0 | 0.6–0.8 × 1.0–3.0 |
Colony appearance | Creamy to brown | Light brown to brownish | Smooth, entire, shiny, white, pink or brown | Raised, convex to umbonate, smooth to rough, entire to irregular |
Catalase | + | + | + | + |
Gram staining | Gram-negative | Gram-negative | Gram-negative | Gram-negative |
Oxidase | − | − | − | − |
Motility | Motile or non-motile | Motile or non-motile | Non-motile | No |
Flagellation | Peritrichous | peritrichous | polar | No |
Oxidation of ethanol to acetic acid | + | + | + | + |
Oxidation of acetic acid to CO2 and water | + | + | − | + |
Oxidation of lactate/acetate | + | + | − | + |
Production of cellulose | − | ± | − | ± |
Growth on 0.35% acetic acid | + | + | + | + |
Growth in the presence of 1% KNO3 | − | − | − | ND |
Growth on methanol | ± | − | − | ND |
Growth in 30% D-glucose | − | ± | ± | ND |
Ketogenesis (dihydroxyacetone) from glycerol | + | + | + | + |
Acid production from: Glycerol D-Mannitol Raffinose Sorbitol | ± − − | + − − − | + + − + | ND − ND − |
Production of DHA from glycerol | ± | ± | + | + |
Production of levan-like polysaccharide from sucrose | ± | − | − | − |
Ubiquinone type | Q9 | Q10 | Q10 | Q10 |
G + C content (mol %) | 50.5–60.3 | 55–67 | 52–64 | 56–64 |
Sources | Flowers, fruits, palm wine, vinegar, kefir | Rhizosphere of coffee plants, roots and stem of sugar cane | Strawberry, grape and spoiled jackfruit and sugar-rich environments | Kombucha, vinegar, wine vinegar |
Different Reproduction Mode of Yeast | Morphology Characteristics of Yeast |
---|---|
Vegetative or asexual reproduction | Budding: new cell is produced on the surface of parent cell and then separate Fission: an asexual cell is produced by a septum grown inward from cell wall to halve the long axis of the cell. Blastoconidiation: a mother cell of stalk-like tubular sterigmata produce a terminal conidium |
Sexual reproduction in ascomycetous yeast | Parent cell-bud conjugation Gametangial conjugation Heterothallism conjugation Conjugation between hyphae |
Sexual reproduction in basidiomycetous yeast | Budding haplophase Dikaryotic hyphal phase or self-spore forming diplophase |
Physiological Test | Biochemical Test |
---|---|
Assimilation of carbon and nitrogen sources | Diazonium Blue B reaction |
Fermentation of carbohydrates | Urease test |
Growth at different temperature | |
Growth in vitamin-free medium | |
Growth in high osmotic pressure condition | |
Starch hydrolysis activity |
Commercial System | Description of Tests and Controls Included in the Kits | Incubation Condition | Accuracy (%) | Reference |
---|---|---|---|---|
API 20 C | 19 carbon assimilation test and 1 control test in 20 strips | 30 °C for 72 h | 98.9 | [88] |
API Candida | 5 carbohydrate and 7 enzymes colorimetric test in 10 strips | 35 °C for 18–24 h | 97.4 | [89] |
API 32 C | 29 assimilation tests (carbohydrate, organic acids, and amino acids); 1 negative control, 1 susceptibility test (cycloheximide) and 1 colorimetric test (esculin) in 32 wells. Includes 63 different species in database | 30 °C for 48 h | 92 | [90] |
Auxacolor system | 13 carbohydrate tests with bromocresol purple, test for cycloheximide resistance and phenoloxidase production in 16 wells. | 37 °C for 48 h | 79.4 | [91] |
RapID Yeast Plus system | 5 carbon assimilation tests and 13 enzymatic hydrolysis substrate tests | 30 °C for 4 h | 96 | [92] |
The Uni-Yeast-Tek (UYT) system | 7 carbon assimilation tests, urease, Nitrate and corn meal with Tween 80 agar | 22–26 °C for 2–10 days | 99.8 | [93] |
MicroScan yeast identification panel | 13 aminopeptidase, 3 carbohydrates, 9 glycosidase, phosphatase and urease tests. | 37 °C for 4 h | 86.9 | [94] |
VITEK 2 YST | 4 aminopeptidase, 25 carbohydrate, esculin, 3 glycosidase, nitrate, 2 nitrogen, 9 organic acid, and urea tests | 35 °C for 18 h | 94.8 | [95] |
Technique | Level | Advantage | Disadvantage |
---|---|---|---|
PCR-RFLP | Species | Rapid, cheap, and easy to set up; suitable for genotyping of AAB isolates | Difficult to identify small insertions and expensive, unable to discriminate closely related species |
DGGE | Species | Rapid and cost-effective; suitable for estimation of AAB diversity | Cannot discriminate closely related species. |
Real-time PCR | Species | Rapid, reliable and quantitative; suitable for comparing microbial abundance. | Complex and expensive |
RAPD | Strain | Quick and simple | Low reproducibility, as the quality and concentration of template DNA influence the results |
ALFP | Strain | Can be used for any DNA samples of any origin, and reveal multiple polymorphic bands in one lane. | Complex and sensitive |
Method | Microorganism | Primer Sequence (5′–3′) | Reference |
---|---|---|---|
16S rRNA, Sanger sequencing | Gluconacetobacter; | 27F, AGAGTTTGATCMTGGCTCAG | [107] |
Komagataeibacter; | 1494R, TGACTGACTGAGGYTACCTTGTTACGACTT | ||
Gluconacetobacter; | fD1, CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG | [29] | |
kombuchae sp. nov.; | rD1, CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC | ||
Acetobacter aceti; | 16S d, GCTGGCGGCATGCTTAACACA | [96] | |
16S r, GCAGGTGATCCAGCCGCA | |||
16S rRNA V1-V3 region, Illumina MiSeq | Bacterial communities | Forward, TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG | [108] |
Reverse, GTVTVGTGGGCTCGGAGATGTGTATAAGAGACAG | |||
16S rRNA V3-V4 region, Illumina MiSeq | Bacterial communities | Forward, CCTACGGGNGGCWGCAG | [102] [109] |
Reverse, GACTACHVGGGTATCTAATCC | |||
16S rRNA, Real-time PCR | Bacterial abundance | 926f, AAACTCAAKGAATTGACGG | [100] |
1062r, CTCACRRCACGAGCTGAC | |||
16S rRNA, T-RFLP | Bacterial communities | 27F, AGAGTTTGATCMTGGCTCAG | [17] |
1525R, AAGGAGGTGATCCAGCC | |||
RAPD | Komagataeibacter spp. | M13, GAGGGTGGCGGTTCT | [104] |
AFLP | Komagataeibacter rhaeticus | A03, GACTGCGTACAGGCCCCG | [110] |
T03, CGATGAGTCCTGACCGAG | |||
REP-PCR | G. oxydans; A. aceti | REPIR-I, IIICGICGICATCIGGC | [105] |
REP2-I, ICGICTTATCIGGCCTAC | |||
ERIC-PCR | G. oxydans; A. aceti | ERIC1R, ATGTAAGCTCCTGGGGATTCAC | [105] |
ERIC2, AAGTAAGTGACTGGGGTGAGCG | |||
Shotgun metagenomic sequencing | Bacterial and fungal communities | [101] [102] |
Method | Microorganism | Primer Sequence (5′–3′) | Reference |
---|---|---|---|
26S rDNA, D1/D2 domain | Brettanomyces/Dekkera; Pichia; B. bruxellensis; | NL1, GCATATCAATAAGCGGAGGAAAAG | [107] [104] |
D. bruxellensis; Hanseniaspora (H.) uvarum | NL4, GGTCCGTGTTTCAAGACGG | [109] | |
26S rDNA, D1/D2 domain | D. bruxellensis; D. anaomala; Z. bailii; H. valbyensis | NL1, GCATATCAATAAGCGGAGGAAAAG | [108] |
NL4, GGTCCGTGTTTCAAGACGG | |||
18S rDNA, D1/D2 domain | Z. kombuchaensis sp. | NS-1, GTAGTCATATGCTTGTCTC | [59] |
NS-8A, CCTTCCGCAGGTTCACCTACGGAAACC | |||
ITS, Illumina MiSeq | Z. bailii | ITS1F, CTTGGTCATTTAGAGGAAGTAA | [102] |
ITS2R, GCTGCGTTCTTCATCGATGC | |||
Real-time PCR | Brettanomyces | Yeast-F, GAGTCGAGTTGTTTGGGAATGC | [100] |
Yeast-R, TCTCTTTCCAAAGTTCTTTTCATCTTT | |||
PCR-ITS RFLP | D. bruxellensis; | ITS1, TCCGTAGGTGAACCTGCGG | [47] |
ITS4, TCCTCCGCTTATTGATATGC |
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Wang, B.; Rutherfurd-Markwick, K.; Zhang, X.-X.; Mutukumira, A.N. Kombucha: Production and Microbiological Research. Foods 2022, 11, 3456. https://doi.org/10.3390/foods11213456
Wang B, Rutherfurd-Markwick K, Zhang X-X, Mutukumira AN. Kombucha: Production and Microbiological Research. Foods. 2022; 11(21):3456. https://doi.org/10.3390/foods11213456
Chicago/Turabian StyleWang, Boying, Kay Rutherfurd-Markwick, Xue-Xian Zhang, and Anthony N. Mutukumira. 2022. "Kombucha: Production and Microbiological Research" Foods 11, no. 21: 3456. https://doi.org/10.3390/foods11213456