Ultrasonically Improved Kombucha Fermentation

Sonication promotes fermentation in ultrasonically fermented foods such as kombucha, kimchi and other fermented vegetables by enhancing mass transfer, disrupting microbial cells, activating enzymes, and improving homogeneity, ultimately leading to accelerated fermentation rates and the production of a superior-quality product. Sonication initiates beneficial changes in bioactive compounds during lactic fermentation increasing the content of nutritional compounds and phytochemicals.

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.

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.

The Benefits of Sonication on Kombucha Fermentation

How does ultrasonic promote the fermentation process and helps to produce better kombucha, vegetable ferments, koji, etc.? Sonication intenisfies fermentation in several ways, which are demonstrated below in the context of fermented kombucha. Kombucha is a fermented beverage traditionally made with sweetened tea and a symbiotic culture of bacteria and yeast (SCOBY). Diluted fruit purees provide a nutrient-rich, flavourful base for kombucha production. Below, you will learn how ultrasonication promotes the production of a

• Increased Mass Transfer: Ultrasonic waves create microscopic cavitation bubbles in the liquid, leading to the formation of microstreaming, liquid jets and turbulences. This agitation enhances mass transfer by increasing the contact between the microorganisms responsible for fermentation and the nutrients in the medium. As a result, nutrients are more efficiently absorbed by the fermenting microorganisms, leading to accelerated fermentation rates.
• Cell Disruption: Sonicators are well-known for their effectiveness of cell lysis and extraction. In food fermentation, sonicators disrupt microbial cell walls, releasing intracellular enzymes and metabolites that can further catalyze fermentation reactions. This disruption enhances the release of flavour compounds, vitamins, and organic acids from the microbial cells, contributing to the flavour complexity and nutrient richness of the fermented product. In ultrasonically-fermented seabuckthorn kombucha, a significantly higher fraction of phenolic compounds can be measured. (cf. Dornan et al., 2020)
• Preparation of Nutrient-Rich Fermentation Substrates: Ultrasonic extraction helps to prepare a fermentation substrate that provides many nutrients in available form for microbial digestion. This means that in ultrasonically treated fermentation substrates (e.g. fruit and vegetable purees) bioactive compounds such as starches and sugars are freed from the intracellular matrix of plant cells. Microbes can readily feed on the substrate, which accelerates and shortens the fermentation process. The same goes for polyphenols, flavonoids and vitamins, which are released from intracellular matrices and contribute to the overall nutritional value of the fermented food or beverages.
• Enhanced Enzyme Activity: Sonication activates or enhances the activity of certain enzymes involved in fermentation processes. For example, it increases the activity of cellulase and amylase, enzymes crucial for breaking down complex carbohydrates into simpler sugars, which are then fermented by the microorganisms present in the kombucha culture.
• Improved Homogeneity: As sonication forces results always in mixing and blending, the ultrasonic treatment ensures better homogenization of the fermentation mixture, resulting in uniform distribution of nutrients and microorganisms throughout the medium. This uniformity promotes consistent fermentation kinetics and the production of a high-quality kombucha product with desirable sensory attributes.

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

Case Study: Ultrasonic Stimulation of Apple Juice 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)

Case Study: 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)

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!

Case Study: Ultrasonically Preserved Kombucha

Ultrasound 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 sonicator 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 the UIP500hdT ultrasonic processor (see picture left). 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.

Case Study: 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.

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

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: