Black Garlic

Added on by Josh Evans.

An old product. Originally from East Asia. Tastes good everywhere.

The cloves start hard, raw, pungent, white, then transform completely – soft, bold, and black, aromatic but not aggressive, like a sort of fruit with beautiful round acidity, notes of balsamic vinegar, molasses, liquorice, tamarind.

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The basic technique is simple – place whole heads in a sealed container and keep at 60˚c for six weeks. One can keep them in for longer and they will mature further, but may also begin to dry out. What is interesting is that the process is not, strictly speaking, fermentative – the transformation is due not to microbial metabolism but in part to enzymatic breakdown (the heat denatures alliinase, the enzyme that converts non-volatile alliin into volatile allicin, the compound responsible for fresh garlic's pungency) and in part to the Maillard Reaction, a cascade of chemical reactions that produce the dark colour and complex, carmelised flavour.[i] The sulfurous compounds may also contribute to its anti-microbial properties in its blackened form.

Foods like this invite a larger discussion about the different degrees of 'fermentation' as a category. A biochemist might stick to the purest technical definition of the anaerobic conversion of glucose into ethanol and carbon dioxide (C6H12O6 → 2 C2H5OH + 2 CO2), while on the other end of the spectrum, a common understanding of fermentation might also include transformations due solely to chemical and enzymatic activity, even though they look, smell, and taste 'fermented' – like black garlic. We and others sit somewhere in the middle, understanding fermentation in the practicable sense as any interaction with microorganisms – bacteria, yeasts, and other fungi – with the purpose of transforming foods.

The principle may be simple, but like any process, the best results lie in obsessing over the details: here, controlling variations in temperature and humidity over time. We are continuing to tweak to discover the best possible product.

In addition to the garlic, we have been experimenting with other alliaceous bulbs. Onions, shallots, it works with them all. Shallots reveal their latent fragrant sweetness; onions intensify into bombs of savouriness; all become tinged with this dark, tart depth.

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These larger bulbs work especially well in broths, a particularly effective way to make a rich, full, dark stock. It also works well in broths with umami-bringing ingredients, like dried aged seaweeds and cured aged meats. And it is satisfying and dramatic with any fermented dairy product – creamy and fleshy, lactic and fruity, white and black.

Some of the cloves we dried and ground to a powder, adding them to salad dressings, sauces, and sprinkled on any number of things. The softer ones we passed through a sieve and pulled into a paste.

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This one is best slathered on crisp toast with skyr, or on its own by the spoonful.

References

[i] Wang, Danan et al. Black Garlic (Allium sativum) Extracts Enhance the Immune System. Medicinal and Aromatic Plant Science and Biotechnology. 4 (1), 37-40. Global Science Books 2010.

Bushifying away the Boar Taint

Added on by Anne Overmark.

posted by Anne Overmark

For the past two months or so I have had the pleasure of playing around with some pork of shady character, a Weber hot smoking grill and some (hopefully) potent microorganisms. My intention with all this? To make a delicious product out of an ingredient many consider repulsively flawed – boar-tainted meat.  

It began with the prospect of a voluntary ban on castration of male piglets within the European Union in the year 2018. Castration is a practice primarily performed to prevent boar taint, the unpleasant odour and flavour that may occur in meat from uncastrated males (Lunde et al. 2013), and which is commonly believed to be caused by the two compounds skatole (3-methylindole) and androstenone (5α-androst-16-en-3-on) (Stolzenbach et al.  2009). However, due to animal welfare issues, this practice is expected to be voluntarily abandoned (Lunde et al 2013 and Font-i-Furnols 2012), which could well lead to an increase in boar-tainted meat that has limited use. There is a resulting interest shared by industry and the consumer to compensate for the off-odours and off-flavours caused by skatole and androstenone and to improve the sensory acceptability of products made of meat from uncastrated male pigs. According to Luckow et al. (2006) masking is one technique that has been used to reduce the perception of aversive odours and flavours in foods through the addition of different flavour compounds to different cuts of pork containing various amounts of skatole (Lunde et al. 2008 ; Stolzenbach et al. 2009 ; Lunde et al. 2013). Nordic Food Lab has undertaken some previous experiments in applying processing methods similar to those used in the production of Katsuobushi to pork. Inspired by this work I will investigate how cooking, drying, smoking, and fermenting will affect the flavour and odour of boar-tainted meat.

Traditionally Katsuobushi, a dried fermented product, is processed from bonito/skipjack tuna (Katsuwonus; Katsuo in Japanese), the processing of which dates back to the Edo period (1603-1868) (Fujita 2009). Today Katsuobushi is processed in two Japanese coastal cities, Tosa and Makurazaki, in which around 70 small family businesses process the fresh bonito into Katsuobushi (Mouritzen and Styrbæk 2011). Katsoubushi is divided into two main categories: Arebushi which is not fermented and Karebushi which is (Mouritzen and Styrbæk 2011). The bonito goes through four steps – cutting, cooking, smoking and drying/fermenting – before it becomes Kastuobushi (McGee 2004, Mouritzen and Styrbæk 2011).  At first the bonito is cut into two filets, which are simmered in salted 90⁰C water for about one hour. After cooking the bones and skin are removed. The filets are then smoked in a hot smoker for at least 6 hours for 14 consecutive days, resulting in a product called Arabushi which contains 40% of the original water content and is covered by a tarlike layer (Mouritzen and Styrbæk 2011). Arabushi is used in foods but the filets can also be further processed into the finer and more palatable Karebushi. In the further processing of Karebushi, the tarlike layer is removed and the filets are left to dry in the sun for a few days. To remove even more of the remaining moisture the filets are inoculated with different moulds. The literature is not consistent with regard to exactly which ones, but the species Aspergillus glaucus, Aspergillus melleus, Aspergillus repens, Aspergillus candidus and Pencillium glaucum are all mentioned (Mouritzen and Styrbæk 2011, Hesseltine 1983, Nout 2007), with Aspergillus glaucus mentioned most often. The inoculated filets are placed in a room in which they undergo fermentation for about two weeks (McGee 2004). After two weeks the fish is brought out to dry in the sun for a day or two and the mould is scraped off. This moulding and drying process is repeated several times, and at the end of the processing, which lasts three to five months, the meat will, when struck, “sound like a resonant piece of wood” (McGee 2004).

I started out with 9 pork filets (Longissimus dorsi). Three came from the meat supplier, so it did not contain skatole or androstenone above the threshold values (in pure solutions, .20-.25ppm and .5-1.0ppm respectively (Tørngren 2010)) and the remaining six were selected at the slaughterhouse in Ringsted. The skatole levels (ppm) in the final six were: .34, .34, .37, .50, .52 and .58 (measured in the back fat of the carcass). The filets were stuffed into nets (like those used for hams) then steamed at 100⁰C until a core temperature of 70⁰C was reached.

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The filets were then left to dry before they were hot-smoked with wood chips made of apple, mesquite and hickory wood for 4 hours. The smoking was repeated the following day before the filets were left in a cold and dry place for three days. The initial processing steps took place at Nordic Food Lab, while the fermentation and final drying stages are being carried out in a climate-controlled chamber at the Danish Meat Research Institute (DMRI) in Roskilde. As previously mentioned mostly Aspergillus species are used in the traditional processing of Katsuobushi, but due to the constraints of EU food safety regulation the filets were inoculated with Pencillium nalgiovense strains, which are approved to be used on meat intended for consumption. Three packages of freeze-dried spores were supplied by Chr. Hansen and Danisco. The strains are probably very similar with regard to how they will affect the flavour and odour; however I decided to try out all three packages and inoculated three filets with each different strain. The spore solutions were prepared according to the instructions on the packages. I made 1L of each in sterile plastic bags and bathed the filets in their respective inocula. One day prior to inoculation the climate chamber was set to have a relative humidity (RH) of 95%, a temperature of 22⁰C and an air flow of 25% of maximum (It is very important that the spores get optimal conditions, to ensure a fast onset of the fermentation). After inoculation the filets were hung with space between them in the climate-controlled chamber for four days, and when I opened the door after the weekend, a nice white layer had covered them completely. Three of the filets had more fuzz compared to the others, but it was likely a characteristic specific to one strain.

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The processing of the boar-tainted meat is at the time of writing in its final, but most time-consuming, step: the drying process. I have lowered the relative humidity by 10% per week, and it is now down to 75%. The temperature is lowered to 15⁰C and I plan to keep it there until it is finished. I have weighed the meat before and after each processing step, to be able to calculate the moisture loss and predict the risk of potential growth of contaminating pathogenic microorganisms, and I certainly hope it is safe to eat as I am very excited to taste the bushi in about a month.

To be continued when new data becomes available.

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References:

Fujita, C., 2009: “Dried Bonito”. The Tokoyo Foundation. http://www.tokyofoundation.org/en/topics/japanese-traditional-foods/vol.-15-dried-bonito. Found December 5th 2012.

Hesseltine, C.W., 1983. ” Microbiology of Oriental Fermented Foods”. Ann. Rev. Microbiol. 1983. 37:575

Luckow. T., Sheehan. V., Fitzgerald. G. and Delahunty. C. (2006) Exposure, health information and flavour-masking strategies for improving the sensory quality of probiotic juice. Appetite. Vol 47. pp. 315-323.

Lunde. K., Egelandsdal. B., Choinski. J., Flåtten. A. and Kubberød. E. (2008) Marinating as a technology to shift sensory thresholds in ready-to-eat entire male pork meat.  Meat Science. Vol. 80. pp. 1264-1272. 

Lunde. K., Skuterud. E., Lindahl. G., Hersleth. M. and Egelandsdal. B. (2013) Consumer acceptability of differently processed bacons using raw materials from entire males. LWT- Food Science and Technology. Vol 51. pp. 205-210.

McGee, H., 2004: “McGee on Food & Cooking an encyclopedia of kitchen science, history and culture”.  Hodder and Stoughton

Mouritsen, O.G. and Styrbæk, K., 2011: “ Umami – Gourmetaben & den femte smag”.

Nout, R. M. J., 2007: ” Food Mycology- A multifaceted approach to fungi and food”. Chap. 17. Mycology. Vol. 25. CRC Press.

Stolzenbach, S., Lindahl, G., Lundström, K., Chen, G. and Byrne, D. (2009). Perceptual masking of boar taint in Swedish fermented sausages. Meat Science. Vol. 81. Pp. 580-588.

Tørngren, A.M. (2012). Litteraturstudie- Anvendelse af lugtende hangrisekød. Danish Meat Research Institute. Project nr. 1378600.

Kombucha: a tasty Symbiotic Culture of Bacteria and Yeasts

Added on by Jonas Astrup Pedersen.

posted by Jonas Astrup Pedersen

Kombucha, also known as Kargasok Tea, Tea Fungus, Haipao and Manchurian Mushroom, is a fermented beverage dating back several thousand years in the East. More recently, it has become popular in the West, specifically in ‘New Age’ circles (Battikh et al., 2012; Jarrell et al., 2000; Greenwalt et al., 2000). Tea fungus initially originated in China in 220 BCE during the Tsin Dynasty and prized as the ‘Divine Che’. The name ‘Kombucha’ seems associated with Doctor Kombu, who is said to have brought the 'tea fungus' from Korea to Japan in 414 CE (Dufresne and Farnworth, 2000).

Increasing interest in Kombucha products is linked to their supposed therapeutic benefits, ranging from curing cancer and AIDS to enhancing weight loss, as well as demonstrating interesting sensory properties (Dufresne and Farnworth, 2000; Teoh et al., 2004). Although several of these claims are not proven, Kombucha beverages exerts antimicrobial activity against Salmonella typhimurium, Staphylococcus aureus, Helicobacter pylori, (Greenwalt et al., 1998), Shigella sonnei, Salmonella enteritidis and Escherichia coli (Greenwalt et al., 1998; Sreeramulu et al., 2001). Furthermore, Kombucha tea ingestion by mice contributed significantly to both life elongation and weight gain inhibition (Hartmann et al., 2000).

The beverage is typically made with black tea, sweetened with 5 to 15% of sucrose, and set to ferment at room temperatures for 10-12 days with a culture popularly known as a ‘tea fungus’, Medusomyces gisevii (Anken and Kappel, 1992; Jayabalan et al., 2010). Inoculation of new batches uses about 10% of Kombucha from a previous batch. The brewing vessel is covered with a clean cotton cloth to keep out debris while allowing aeration (Greenwalt et al., 2000). A schematic description of Kombucha production is seen in Figure 1.

Figure 1: Schematic overview of producing Kombucha

Figure 1: Schematic overview of producing Kombucha

Kombucha is the expression of a symbiotic growth of bacteria such as Acetobacter xylinum, A. xylinoides, A. aceti, A. pasteurianus, Bacterium gluconicum (Sreeramulu et al., 2000; Dufresne and Farnworth, 2000) and yeasts like Schizosaccharomyces pombe, Kloeckera apiculata, Saccharomycodes ludwigii, Saccharomyces cerevisiae, Zygosaccharomyces bailii, Brettanomyces bruxellensis, B. lambicus, B. custersii and Pichia species (Dufresne and Farnworth, 2000). Though the fungus-like cellulosic matrix produced by especially Acetobacter xylinum might look like a fungus (Mo et al., 2008), ‘tea fungus’ is rather misleading since the ‘tea fungus’ is in fact only a physical manifestation of the yeast and bacteria symbiosis (Sreeramulu et al., 2000). The floating jelly-like membrane, called a zoogleal mat, is where the cell mass of the bacteria and yeasts are attached (Jayabalan et al., 2010). The cellulose is a secondary metabolite of the fermentation, similar in structure to a ‘mother of vinegar’ (Jayabalan et al., 2010). Within the cellulose network, investigations have shown the Kombucha colony to be arranged in bands and layers (Anken and Kappel, 1992), see Picture 4. The composition and exact diversity of the microbiological presence depends on the source of the Kombucha culture (Sreeramulu et al., 2000).

As yeast cells hydrolyze sucrose into glucose and fructose, producing ethanol and carbon dioxide as metabolites, acetic acid bacteria converts glucose into gluconic acid and fructose into acetic acid (Reiss, 1994; Loncar et al., 2006). The primary metabolites of ethanol and acetic acid behave as catalyzing agents; yeast are stimulated to produce ethanol by acetic acid, whereas ethanol stimulates the growth of acetic acid bacteria and their production of acetic acid (Liu et al., 1996). Fructose is utilized to a lesser degree and remains part of the fermented liquid (Greenwalt et al., 1998). The synthesis of complex B vitamins and folic acids has also been reported during the fermentation process (Bauer-Petrovska and Petrushevska-Tozi, 2000). Additionally, the organic acids produced throughout the fermentation and the corresponding decrease in pH value prevent the symbiotic culture from becoming contaminated by undesirable microorganisms not contained in the tea fungus (Greenwalt et al., 1998; Mo et al., 2008).

The properties and composition of the final product depends on the initial substrates, to geographical and climatic conditions, as well as the locally-specific types of wild yeast and bacteria present (Bauer-Petrovska and Petrushevska-Tozi, 2000). To obtain beneficial attributes and antimicrobial activity against a range of pathogenic bacteria, Greenwalt et al. (1998) recommends consumption of Kombucha containing 33 g/L total acids, 7 g/L acetic acid. Usually, the pH of a fermented Kombucha is around 2.5, regarded by the food industry as a high-acid food since a pH of 4.0 prevents growth of most organisms linked with spoilage (Greenwalt et al., 2000).

Experiments

Herbs or wood and boiling water (1 L each) were added to separate containers, closed with a lid and left to infuse at room temperature with different infusing times for optimal flavour intensity. These included dried yarrow flowers (1% w/v, 10 min.), juniper wood (5% w/v, 1 hour), dried chamomile (1% w/v, 10 min.), dried lemon verbena (1% w/v, 10 min.), dried woodruff (1% w/v, 10 min.) and dried cèpes (5% w/v, 12 hour). Dried kelp (2.3% w/v, 1 hour) was sealed in a vacuum bag and treated sous vide at 60 °C. To all solutions were added 50 g of sucrose.

Picture 1-3:  herbs, cepes and wood extraction (top); straining tea (bottom left); inoculated yarrow flower tea (bottom right).

Picture 1-3:  herbs, cepes and wood extraction (top); straining tea (bottom left); inoculated yarrow flower tea (bottom right).

The Kombucha mothers were carefully cut into approximately equal sizes and added to the teas. Additionally, 100 mL (10% v/v) of the liquid medium (tea kvass) were also added. Containers were covered with a cloth and set into a closed cabinet at an ambient temperature of approximately 21 ± 2 °C.

Table 1: pH of the different infusions at initial stage and after 12 days of fermentation

Table 1: pH of the different infusions at initial stage and after 12 days of fermentation

Based on sensory evaluation among employees at Nordic Food Lab (NFL) during the period of fermentation and at day 12, lemon verbena (Aloysia triphylla) was selected as a basis for further elaboration. The absence of data for cepe and carrot is due to loss of samples to mold, most likely because of too high initial pH.

To produce sufficient quantity of the lemon verbena Kombucha for further investigation, a batch of 17 L of tea was brewed: dried lemon verbena (1% w/v, 10 min.) was steeped in boiling water, to which was added 50 g of sucrose per liter. The batch was inoculated with tea kvass (0.95 mL) from the previous batch. The container was put into an incubator-box to control fermentation temperatures, which measured in the range of 27 ± 2 °C.

Picture 4: the cellulose network, zoogleal mat (Kombucha mother) produced by Acetobacter xylinum.

Picture 4: the cellulose network, zoogleal mat (Kombucha mother) produced by Acetobacter xylinum.

Lemon verbena Kombucha

The herb mentioned as lemon verbena (Aloysia triphylla (L’Hérit.) Britt. Syn. Lippia citriodora) belongs to the family of Verbenáeae (in Danish: jernurt-familien) (Vogel et al., 1999). It is well known for the pleasant odour of its leaves, comparable to that of a lemon. Responsible for its aromatic properties are essential oils found in concentrations of 0.4% (Montes et al., 1973) to 1.2% (Vogel et al., 1999). Its pronounced lemon-like odour is due to the chemical compound citral found in concentration of lemon verbena oils between 11% and 54% (Montes et al., 1973; Vogel et al., 1999).

Lemon verbena, also called cedrón in its countries of origin, is a shrub native to Peru, Chile and Argentina where it is cultivated for domestic consumption as an herb tea (Vogel et al., 1999). It was brought to Europe during the 18th century and grown as potted plants due to its high sensitivity to cold (Vogel et al., 1999). Despite its origin in South America, lemon verbena has caught the attention of chefs and is now found in many Nordic kitchens and dishes.

In cooking circles, an often-heard misunderstanding of its name is the simple use of (in Danish) jernurt. Jernurt refers merely to the family Verbenáeae, which contains some 25-34 genera and 500-1200 species comprising a great variety of small trees, lianas, shrubs and herbs (Yuan et al., 2010).

In an attempt to speed up the fermentation process, aeration was tested on four brews. An aquarium pump (AM-TOP model CR10) was connected to the containers during the entire fermentation process. Different kinds of sugar, either 'alone' or in combination, were tested as different energy sources for the yeast and bacteria. This was done with the purpose of observing and detect possible variation in sensory qualities and pH of end products. Teas were prepared as in the preliminary trials: herb extraction (1% w/v, 10 min.), added sugar (5% w/v) and finally inoculation with tea kvass (10% v/v) at 21 ± 2 °C, see Table 2 and Table 3. Where fructose is stated, Danish honey from Søborg was used.

Table 2: pH of different infusions at initial stage and after 7 days of fermentation

Table 2: pH of different infusions at initial stage and after 7 days of fermentation

Table 3: pH of different infusions at initial stage and after 7 days of fermentation

Table 3: pH of different infusions at initial stage and after 7 days of fermentation

Results

With the aerated batches, enhancing the fermentation speed did not seem to succeed in favouring the acetic acid bacteria. Of the four batches, none were as balanced, as complex in flavor, or as refreshing as Kombucha can be. Although Kombucha is acidic, this tartness was not satisfying when complementary aroma was absent or expressed in a flat manner.

Testing different sugars revealed great difference in end product. Some Kombuchas turned out almost vinegar-like and not appropriate for a soft drink, though these could have interesting applications for culinary exploration and use. By far the most aromatic and interesting Kombucha developed in this experiment, contained sucrose as the sugar substrate. It expressed herby notes as well as an interesting ginger-like association, well balanced, pleasantly acidic and complex.

Alongside these small-batch trials, a continuous batch has served as staff Kombucha. Sweetened lemon verbena tea has been added, throughout the project, to a large vessel used during the process, while Kombucha has been tapped and enjoyed. The Kombucha turned out very tasty in expression: fizzy, refreshing, and especially delicious when served ice-cold. This encourages further investigation into continuous fermentations with e.g. higher levels of inoculum to amount of sweetened tea, as well as second fermentations in terms of added juice or other flavourfull sugar containing liquids, is a path believed worth investigating. KOMBOOOUCHA!

Literature

[1] ANKEN, R. H. & KAPPEL, T. 1992. Histochemical and anatomical observations upon the tea fungus, Liége, Belgique, Vaillant-Carmanne.
[2] BATTIKH, H., BAKHROUF, A. & AMMAR, E. 2012. Antimicrobial effect of Kombucha analogues. LWT - Food Science and Technology, 47, 71-77.
[3] BAUER-PETROVSKA, B. & PETRUSHEVSKA-TOZI, L. 2000. Mineral and water soluble vitamin content in the Kombucha drink. International Journal of Food Science and Technology, 35, 201-205.
[4] DUFRESNE, C. & FARNWORTH, E. 2000. Tea, Kombucha, and health: a review. Food Research International, 33, 409-421.
[5] GREENWALT, C. J., LEDFORD, R. A. & STEINKRAUS, K. H. 1998. Determination and Characterization of the Antimicrobial Activity of the Fermented Tea Kombucha. LWT - Food Science and Technology, 31, 291-296.
[6] GREENWALT, C. J., STEINKRAUS, K. H. & LEDFORD, R. A. 2000. Kombucha, the fermented tea: Microbiology, composition, and claimed health effects. Journal of Food Protection, 63, 976-981.
[7] HARTMANN, A. M., BURLESON, L. E., HOLMES, A. K. & GEIST, C. R. 2000. Effects of chronic kombucha ingestion on open-field behaviors, longevity, appetitive behaviors, and organs in c57-bl/6 mice: a pilot study. Nutrition, 16, 755-761.
[8] JARRELL, J., CAL, T. & BENNETT, J. W. 2000. The Kombucha consortia of yeasts and bacteria. Mycologist, 14, 166-170.
[9] JAYABALAN, R., MALINI, K., SATHISHKUMAR, M., SWAMINATHAN, K. & YUN, S.-E. 2010. Biochemical characteristics of tea fungus produced during kombucha fermentation. Food Science and Biotechnology, 19, 843-847.
[10] LIU, C. H., HSU, W. H., LEE, F. L. & LIAO, C. C. 1996. The isolation and identification of microbes from a fermented tea beverage, Haipao, and their interactions during Haipao fermentation. Food Microbiology, 13, 407-415.
[11] LONCAR, E., DJURIC, M., MALBASA, R., KOLAROV, L. J. & KLASNJA, M. 2006. Influence of Working Conditions Upon Kombucha Conducted Fermentation of Black Tea. Food and Bioproducts Processing, 84, 186-192.
[12] MO, H., ZHU, Y. & CHEN, Z. 2008. Microbial fermented tea - a potential source of natural food preservatives. Trends in Food Science & Technology, 19, 124-130.
[13] MONTES, M., VALENZUELA, L., WILKOMIRSKY, T. & ARRIVÉ, M. 1973. Sur La Composition De L'Essence D'Aloysia triphylla ("Cedron"). Planta Med, 23, 119-124.
[14] REISS, J. 1994. Influence of different sugars on the metabolism of the tea fungus. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung, 198, 258-261.
[15] SREERAMULU, G., ZHU, Y. & KNOL, W. 2000. Kombucha Fermentation and Its Antimicrobial Activity. Journal of Agricultural and Food Chemistry, 48, 2589-2594.
[16] SREERAMULU, G., ZHU, Y. & KNOL, W. 2001. Characterization of antimicrobial activity in Kombucha fermentation. Acta Biotechnologica, 21, 49-56.
[17] TEOH, A. L., HEARD, G. & COX, J. 2004. Yeast ecology of Kombucha fermentation. International Journal of Food Microbiology, 95, 119-126.
[18] VOGEL, H., SILVA, M. L. & RAZMILIC, I. 1999. Seasonal flcutuation of essential oil content in lemon verbena (Aloysia triphylla), Mendoza, Agentina
[19] YUAN, Y.-W., LIU, C., MARX, H. E. & OLMSTEAD, R. G. 2010. An empirical demonstration of using pentatricopeptide repeat (PPR) genes as plant phylogenetic tools: Phylogeny of Verbenaceae and the Verbena complex. Molecular Phylogenetics and Evolution, 54, 23-35.