Ultrasonically Improved Kombucha Fermentation

Kombucha is a fermented beverage containing tea, sugar, bacteria, yeast and often a little amount of juice, fruit or spices as flavouring. Kombucha as well as fermented juice and vegetable juices are known for having positive effects on health, strengthening the microbiota and immune system. Controlled ultrasonication improves the production of kombucha and other fermented beverages in various ways: Ultrasound can stimulate yeast and bacteria growth during fermentation; extract polyphenols, flavonoids and flavours from fruits, vegetable and herbs; and also applied as non-thermal pasteurization method for microbial reduction before packaging. Hielscher ultrasonicators are precisely controllable and can deliver the most suitable ultrasonic intensity for each treatment step in the production of fermented beverages.

Kombucha and Fermented Beverages

Kombucha is produced by fermenting sugared tea using a “Symbiotic Culture Of Bacteria and Yeast” (SCOBY), also commonly called a “mother” or “tea mushroom”. The variety and ratio of microbial populations in a SCOBY can vary quite significantly. The yeast component generally includes Saccharomyces cerevisiae, along with other species of Zygosaccharomyces, Candida, Kloeckera/Hanseniaspora, Torulaspora, Pichia, Brettanomyces/Dekkera, Saccharomyces, Lachancea, Saccharomycoides, Schizosaccharomyces, and Kluyveromyces; the bacterial component almost always includes Komagataeibacter xylinus (formerly Gluconacetobacter xylinus), which ferments alcohols produced by the yeasts into acetic and other acids, increasing the acidity and limiting ethanol content.
Similarly, other fermented beverages such as fermented fruit and vegetable juices are inoculated with bacteria and yeast.
A treatment with ultrasound can improve fermentation efficiency and quality features of the fermented beverage including nutrient content and flavour.

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Ultrasonic bioreactor with 2000 watts ultrasonic probe for improved fermentation processes, e.g. for the production of kombucha and other fermented beverages.

Ultrasonic bioreactor for the stimulation of bacterial and yeast growth. Ultrasonic stimulation of microorganisms results in improved fermentation processes.

How Does Ultrasound Improve Kombucha Fermentation?

  • More efficient fermentation
  • Extraction of nutritional compounds (e.g., polyphenols, flavonoids etc.)
  • Extraction of flavour compounds
  • Preservation, microbial stabilization

Ultrasonically Intensified Kombucha Fermentation

Ultrasound waves are well known to stimulate the growth of bacteria and yeast. A controlled mild sonication of kombucha cultures (SCOBY, also known as tea mushroom, tea fungus, or Manchurian mushroom) can therefore promote the fermentation process and lead to higher kombucha yields in an accelerated fermentation time.
Ultrasonically-stimulated fermentation shows enhanced membrane permeabilization and thereby increased mass transfer. The sonomechanical treatment by ultrasound waves perforates the cell walls and plasma membranes of microorganisms (a process called sonoporation). Some cells might be even ruptured. These disrupted cells release growth promoting factors such as vitamins, nucleotides, amino acids and enzymes that could stimulate the growth of cellularly intact as well as membrane compromised bacteria.
Ultrasonic treatment prior fermentation as well as in the lag and log phases showed the most prominent effects on bacterial growth stimulation.

Ultrasonic treatment of microorganisms can stimulate their growth, thereby improving subsequent fermentation processes. (graph adapted from Michal Komoniczak)

Ultrasonic stimulation of microbial growth is most effective when ultrasound is applied prior fermentation or in the lag and log phases.

Ultrasonic Stimulation of Apple Juice Fermentation

Probe-type ultrasonicator for fruit and vegetable juice extraction and fermentation.Research showed that ultrasonic treatment at lag and logarithmic phases during the fermentation of apple juice promoted microbial growth and intensified biotransformation of malic acid to lactic acid. For example, after sonication at lag phase for 0.5 h, microbial count and lactic acid content in the ultrasound-treated samples at 58.3 W/L reached 7.91 ± 0.01 Log CFU/mL and 133.70 ± 7.39 mg/L, which were significantly higher than that in the non-sonicated samples. Moreover, ultrasonication at lag and logarithmic phases had complex influences on the metabolism of apple phenolics such as chlorogenic acid, caffeic acid, procyanidin B2, catechin and gallic acid. Ultrasound could positively affect the hydrolysis of chlorogenic acid to caffeic acid, the transformation of procyanidin B2 and decarboxylation of gallic acid. The metabolism of organic acids and free amino acids in the sonicated samples was statistically correlated with phenolic metabolism, implying that ultrasound may benefit phenolic derivation by improving the microbial metabolism of organic acids and amino acids. (cf. Wang et al., 2021)

Ultrasonically Improved Soy Milk Fermentation

The research team of Ewe et al. (2012) investigated the effects of ultrasound on metabolic efficiency of strains of lactobacilli (Lactobacillus acidophilus BT 1088, L. fermentum BT 8219, L. acidophilus FTDC 8633, L. gasseri FTDC 8131) during soymilk fermentation. It was observed that the ultrasonic treatment permeabilized the cellular membranes of the bacteria. The permeabilized cellular membranes resulted in an improved nutrient internalization and subsequent growth enhancement (P ≺ 0.05). Higher amplitudes and longer durations of the sonication treatment promoted growth of lactobacilli in soymilk, with viable counts exceeding 9 log CFU/mL. The intracellular and extracellular β-glucosidase specific activities of lactobacilli were also enhanced (P ≺ 0.05) by ultrasonication, leading to increased bioconversion of isoflavones in soymilk, particularly genistin and malonyl genistin to genistein. Results from this study show that ultrasound treatment on lactobacilli cells promotes (P ≺ 0.05) the β-glucosidase activity of cells for the benefit of enhanced (P ≺ 0.05) isoflavone glucosides bioconversion to bioactive aglycones in soymilk. (cf. Ewe et al., 2012)

Ultrasonication perforates cellular membranes of lactobacilli , which promoted nutrient uptake and growth of lactobacilli in soymilk. This resulted in enhanced b-glucosidase enzyme activities upon fermentation.

Scanning electron micrographs of lactobacilli without treatment (A) and lactobacilli treated with ultrasound at 60% amplitude for 3 min (B). Circles showing ruptured cells and cells with pores.
(Study and images: ©Ewe et al., 2012)

Extraction of Nutritional Compounds and Flavours in Kombucha and Fermented Beverages

Fermented tea, juice and vegetable beverages, e.g. fermented apple or mulberry juice or fruit infused kombuchas, benefit flavour- and nutrient-wise significantly from an ultrasound treatment. The ultrasound waves disrupt cellular structures of plant materials and release intracellular compounds such as flavours, polyphenols, antioxidants and flavonoids. At the same time, ultrasonic homogenization provides a uniformly dispersed and emulsified beverage preventing phase separation and offering an appealing appearance for consumers. You can see an example of an ultrasonically treated sea buckthorns berry kombucha without phase separation in comparison to an untreated version below.
Read more about ultrasonic flavour and nutrient extraction!

High-intensity ultrasonication is introduced into biochemical reactors in order to improve fermentation processes.

Ultrasonic bioreactor with 4x UIP4000hdT for improved fermentation processes

Ultrasound for Kombucha Preservation

Ultrasonic inline homogenizer for juice, dairy and liquid egg pasteurizationUltrasound treatment can influence microbes by either stimulate or inactivate them. Also enzymes are impacted by sonication: Ultrasound can change characteristics of enzymes, substrates and their reactions. These effects of low-frequency ultrasound are used in food processing as an non-thermal alternative to pasteurize food and beverages. Sonication offers the advantage of precise control over the process parameters such as amplitude, time, temperature, and pressure, which allows for a targeted inactivation of microorganisms. The inactivation of the microbial load in kombucha and fermented beverages allows to increase shelf-life and product stability. A reduction of microbes and enzymes facilitates commercial distribution due to an prolonged shelf-life of the final product. Ultrasonication is a non-thermal pasteurization method, which is used already in commercial food processing such as the pasteurization of juices. Especially at higher amplitudes, ultrasound inactivates bacteria and yeast by damaging the cell walls. This results in a slower or stopped microbial growth. For instance, Kwaw et al. (2018) investigated the ultrasonic as non-thermal pasteurization strategy for lactic acid-fermented mulberry juice. Ultrasonically treated fermented mulberry juice had a higher content of phenolic compounds (1700.07 ± 2.44 μg/mL) than the control, an untreated fermented mulberry juice. “Among the individual non-thermal treatments, ultrasonication caused a significant (p < 0.05) upsurge in the phenolic and antioxidant properties of the lactic acid-fermented mulberry juice compared to the pulsed light treatment.” (Kwaw et al., 2018) Whilst kombucha is a beverage known for its life cultures, a controlled reduction of microbes can be used in order to prolong the shelf-life of commercially distributed kombucha beverages. Regular thermal pasteurization kills all the living yeasts and bacteria which are normally present in kombucha and are one of the main factors for its health-promoting effects. Ultrasonic pasteurization is a non-thermal preservation method, which can be used to either reduce microbial counts or eliminate microorganisms completely. This means that commercial producers can apply ultrasound at lower amplitudes and for shorter periods in order to lower the number of bacteria and yeast without eliminating completely. Thereby, life cultures are still present in the kombucha, although at lower numbers so that shelf life and storage times are improved.

Scientifically Proven Results in Ultrasonically-Treated Kombucha

Dornan et al. (2020) investigated the effects of low-frequency ultrasound on kombucha made with seabuckthorn berries using the ultrasonic processor UIP500hdT. The research team could demonstrate multiple benefical effects of ultrasonication on the sea buckthorn berry preparation and subsequent kombucha fermentation.

Ultrasonic Sea Buckthorn Berry Extraction

Sonication of fresh whole sea buckthorns berries (also known as sanddorne; H. rhamnoides cv. Sunny) were puréed using a Vitamix blender for 2 min. A volume of dH2O equal to 30% of the original purée volume was added and blended. Ultrasound (90 W, 20 kHz, 10 min) was applied to 200 mL of the diluted purée using a UIP500hdT ultrasonic processor (Hielscher Ultrasonics, Germany). Treatment time was chosen to optimization of nutrients and maintain the sample in fresh-like state. The results of ultrasonic extraction demonstrates a significant (P ≺ 0.05) increase by 10% extraction yield from pulp (from 19.04 ± 0.08 to 20.97 ± 0.29%) and by 7% for seed (from 14.81 ± 0.08 to 15.83 ± 0.28%). This increase in oil yield highlights the functionality of sonication as an efficacious and green technology to maximize raw material value. Ultrasonic extraction from sea buckthorns berries resulted in higher oil yield and reduced processing time, power consumption, and avoidance of hazardous solvents.

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Ultrasonic Homogenization of Sea Buckthorn Berry Kombucha

The ultrasonically treated sea buckthorns (sanddorne) berry kombucha showed a significantly improved stability of the product. By day 21 of storage, the sonicated berry kombucha remained homogenous. The fact that no syneresis was observed in sonicated berry kombucha for the whole study (21 days, see picture below) shows that ultrasound alone is an effective emulsification technique able to produce product stability and to prevent phase separation.

Ultrasonication improves the stability of seabuckthorne berry kombucha.

Seabuckthorn puree (P) without and with ultrasound treatment (P+US) on day 0 and 21.
Study and picture:©Dornan et al., 2020.

Ultrasonication to Stop Fermentation

Four samples of kombucha were made: K (kombucha), K+US (kombucha + ultrasound), K+S (kombucha + sucrose), and K+S+US (kombucha + sucrose + ultrasound). All samples were prepared using 200 mL of sea buckthorns puree (P) or P+US and 12.5 g of SCOBY. K consisted of P and SCOBY. K+US consisted of P+US and SCOBY. K+S consisted of P, 15.0 g of sucrose and SCOBY. K+S+US consisted of P+US, 15.0 g of sucrose and SCOBY. All samples were left to ferment in a dark place at room temperature for five days. A second sonication treatment (90 W, 20 kHz, 10 min) was applied to K+US and K+S+US to halt fermentation on day 5.

Ultrasonic Preservation Effects on Kombucha

In sea buckthorns kombucha, sonication decreased initial microbial load by 2.6 log CFU/mL, thereby halting the fermentation process at a selected time in order to prevent over-fermentation. Furthermore, the controlled microbial reduction helps to increase the shelf-life and stability of final product, which facilitates the commercial distribution of kombucha.
Read more about ultrasonication as non-thermal juice pasteurization method!

Overall Results in Ultrasonically Treated Kombucha

Ultrasonication decreased initial microbial load by 2.6 log CFU/mL, increased ORAC value by 3% and increased water solubility index (WSI) by 40% (from 6.64 to 9.29 g/g) without syneresis. Results from this study suggest that application of ultrasonication can enhance phenolic functionality during fermentation and is capable of decreasing syneresis, increasing oil yield, decreasing microbial load, and increasing ORAC with minimal loss of nutritional quality. (cf. Dornan et al., 2020)

Ultrasonic Equipment for Improved Kombucha Brewing

Hielscher Ultrasonics designs, manufactures and distributes high-performance ultrasonicators, ultrasonic bioreactors and accessories for improved fermentation, extraction, and pasteurization processes used in food & beverage manufacturing. Hielscher ultrasonic food processing systems are used for manifold applications being a safe, reliable and cost-efficient technology to produce high-quality foods and beverages. Installation and operation of all Hielscher ultrasonic processors is simple: They requires only little space, can be easily retrofitted into existing processing facilities.
Hielscher Ultrasonics is long-experienced in the application of power ultrasound in the food & beverage industry as well as many other industrial branches. Our ultrasonic processors are equipped with easy-to-clean (clean-in-place CIP / sterilize-in-place SIP) sonotrodes and flow-cells (the wet parts). Hielscher Ultrasonics’ industrial ultrasonic processors can deliver very high amplitudes. Amplitudes of up to 200µm can be easily continuously run in 24/7 operation. The precise tuning of amplitudes and the option to switch between low and high amplitudes are important to stimulate or inactivate microorganisms. Thereby, the same ultrasonicator can be used to either stimulate microbes increasing fermentation or to inactivate microorganisms for pasteurization.
State-of-the-art technology, high-performance and sophisticated software make Hielscher Ultrasonics’ reliable work horses in your food fermentation process. With a small footprint and versatile installation options, Hielscher ultrasonicators can be easily integrated or retro-fitted into existing production lines.

Process Standardization with Hielscher Ultrasonics

Hielscher ultrasonicators can be remotely controlled via browser control. Sonication parameters can be monitored and adjusted precisely to the process requirements.Food-grade products should be produced in accordance to Good Manufacturing Practices (GMP) and under standardised processing specifications. Hielscher Ultrasonics’ digital extraction systems come with intelligent software, which makes it easy to set and control the sonication process precisely. Automatic data recording writes all ultrasonic process parameters such as ultrasound energy (total and net energy), amplitude, temperature, pressure (when temp and pressure sensors are mounted) with date and time stamp on the built-in SD-card. This allows you to revise each ultrasonically processed lot . At the same time, reproducibility and continuously high product quality are ensured.
Hielscher Ultrasonics’ industrial ultrasonic processors can deliver very high amplitudes. Amplitudes of up to 200µm can be easily continuously run in 24/7 operation. For even higher amplitudes, customized ultrasonic sonotrodes are available. The robustness of Hielscher’s ultrasonic equipment allows for 24/7 operation at heavy duty and in demanding environments.
Please contact us know to learn more about the features and capability of our ultrasonic pasteurization systems. We would be glad to discuss your application with you!

The table below gives you an indication of the approximate processing capacity of our ultrasonicators:

Batch Volume Flow Rate Recommended Devices
1 to 500mL 10 to 200mL/min UP100H
10 to 2000mL 20 to 400mL/min UP200Ht, UP400St
0.1 to 20L 0.2 to 4L/min UIP2000hdT
10 to 100L 2 to 10L/min UIP4000hdT
n.a. 10 to 100L/min UIP16000
n.a. larger cluster of UIP16000

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Ultrasonic high-shear homogenizers are used in lab, bench-top, pilot and industrial processing.

Hielscher Ultrasonics manufactures high-performance ultrasonic homogenizers for mixing applications, dispersion, emulsification and extraction on lab, pilot and industrial scale.

Literature / References

Facts Worth Knowing

Kombucha Fermentation

The term “kombucha” as well as the production process for kombucha is not legally regulated. This means that many fermented drinks are sold as kombucha beverage, but in the traditional sense “kombucha” is a fermented tea beverage. Kombucha is made by adding the kombucha culture into a broth of sugared tea. The sugar serves as a nutrient for the SCOBY that allows the bacteria and yeast to grow in the sugar liquid. The acetic acid bacteria in kombucha are aerobic, meaning that they require oxygen for their growth and activity. During fermentation, biochemical conversion takes place, which converts sucrose into fructose and glucose. Fructose and glucose are subsequently converted into gluconic acid and acetic acid. In addition, kombucha contains enzymes and amino acids, polyphenols, and various other organic acids which vary between preparations. Other specific components include ethanol, glucuronic acid, glycerol, lactic acid, usnic acid, B-vitamins, and vitamin C. The alcohol content of kombucha is usually less than 0.5% since the bacteria strain of Komagataeibacter xylinus converts ethanol into acids (such as acetic acid). However, extended fermentation increases alcohol content. Over-fermentation generates high amounts of acids similar to vinegar. Kombucha beverages have typically a pH value of approx. 3.5.

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Hielscher Ultrasonics manufactures high-performance ultrasonic homogenizers from lab to industrial size.