- Email: [email protected]
Traditional biotechnology for new foods and beverages
Available online at www.sciencedirect.com
Traditional biotechnology for new foods and beverages Jeroen Hugenholtz The food and beverage industry is re-discovering fermentation as a crucial step in product innovation. Fermentation can provide various benefits such as unique flavor, health and nutrition, texture and safety (shelf life), while maintaining a 100% natural label. In this review several examples are presented on how fermentation is used to replace, modify or improve current, artificially produced, foods and beverages and how also fermentation can be used for completely novel consumer products. Address Swammerdam Institute for Life Sciences, University of Amsterdam and Coca-Cola Corporate Research, Mainburger Strasse 19, 84072 Au/ Hallertau, Germany Corresponding author: Hugenholtz, Jeroen ([email protected])
Current Opinion in Biotechnology 2013, 24:155–159 This review comes from a themed issue on Food biotechnology Edited by Elaine E Vaughan and Jeroen Hugenholtz For a complete overview see the Issue and the Editorial Available online 8th February 2013 0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.01.001
Introduction The process of fermentation was traditionally leveraged by the human population to increase the shelf life of perishable agricultural produce such as milk, vegetables and meat. This has resulted in a vast variety of fermented foods and beverages that is still the main part of the human diet in many under-developed countries and in most countries in South-East Asia. In North America and Europe which have extremely efficient and rapid distribution systems and an overall availability of cooling and freezing systems, most of the traditional fermented products, with the exception of fermented dairy (yoghurt and cheese) and meat (sausages) have been replaced by fresh agricultural produce, making the process of fermentation obsolete. This trend away from fermentation seems to have come to a recent stop and is now gradually reversing. More and more people (and food companies) are regaining interest in traditional and more natural foods and there is a growing dislike for the processing and energyinput that is needed to maintain freshness of agricultural crops. In addition, the food and beverage industry is continuously trying to innovate within the constraints of sustainability and naturalness. All these recent develwww.sciencedirect.com
opments are leading to increased interest and activity in fermentation technology by all consumer goods industries, big and small. This trend is not really visible in the scientific literature and (not yet) in the patent literature, so some referencing will be made to company-websites as support of statements made in this contribution.
Fermentation for all-natural foods or beverages Fermentation, nowadays, is all about bringing unique signature flavors and other benefits to consumer products in a 100% natural way. This is done using, mainly, two different approaches. The ‘easiest’ is to turn a traditional, home-grown, product into a large scale process. Examples are the dairy product, kefir [1], which is a traditional liquid fermented dairy product using a mixture of lactic acid bacteria, yeast and fungi for the fermentation of milk, resulting in a yoghurt-like, slightly alcoholic product which has traditionally been consumed by millions of people in, especially, Eastern Europe and supposedly conveys spectacular health benefits to the consumer [2]. The fermentation process is, typically, conducted at home on, or close to, the stove and using small leftovers of previously produced kefir or a small kefir granule purchased at the local grocery store, as inoculum. Only recently, the larger dairy companies have managed to upscale this process to an industrial scale using active and stable starter cultures just as is the daily practice for mainstream dairy products such as yoghurt and cheese. Another example is the traditional drink of Kvass (http:// rbth.ru/articles/2012/06/22/kvass_russias_real_national_drink_15952.html), which is a result of fermentation of kitchen left-overs mainly consisting of bread bits. This fermented product is also traditionally made at home with a final composition depending on the type of left-overs used and the nature of the locally evolved microbial culture. This traditional beverage is now also marketed by larger companies, also outside the originating country, Russia. The main challenge of these companies is to prepare this family drink with a constant quality and, especially, without alcohol. This is basically done by using controlled fermentations with standardized starter cultures and similar fermentation conditions (short, low temperature) as used for non-alcoholic beer production. Several more examples can be found of food and beverage companies launching new or improved fermented products based on traditional biotechnology [3] (http://bycristinaybarra.com/2012/10/27/burnarj-the-firstsparkling-drink-with-andalusian-taste/, http://www.calpis. net/features/story/index.html, www.the-spirit-of-georgia. de, http://www.doehler.com/en/our_products/beverage_ and_dairy_bases/fermented_products) (Figure 1). Current Opinion in Biotechnology 2013, 24:155–159
156 Food biotechnology
Figure 1
Kefiy
Boza
Amazake
Amasi
Fruit vinegars
Tr fe adi r pr me tion o d n t al uc ed ts
Kombucha
Lassi
Kvass
Current Opinion in Biotechnology
Example of traditional fermented beverages from across the world.
A second approach followed by many consumer goods companies is to transfer a successful, large-scale, fermentation process to other substrates. One typical example is, again, kefir, which traditionally is a dairy product, using milk as substrate. The newest development is to use the traditional kefir culture for fermentation of other substrates, such as fruit juice, resulting in products called ‘fruit kefir’ or something similar. Other examples are biological soft drinks such as Bionade (http://dizzyfrinks. com/drink/bionade-holunder-elderberry/) (in Germany, Switzerland, Austria) and Tumult (http://news.softpedia. com/news/Coca-Cola-Comes-Out-with-Tumult-the-SoftDrink-that-Tastes-like-Beer-216150.shtml) (in France) where the microorganisms of the traditional, fermented tea drink Kombucha [3] have been used for fermentation of malt, leading to a non-alcoholic soft drink with unique flavor for the adult market.
percent natural technology to raise vitamin levels in foods and beverages. Numerous examples, especially involving dairy applications, have been presented showing natural enrichment with riboflavin [4], folate [5], vitamin B12 [6], vitamin K2 [7] and sometimes several of these vitamins, simultaneously [8,9]. The latest developments in this area are discussed in another chapter of this Special issue [10].
In addition to offering unique sensory sensations, as the label on the package or the advertisements will most likely tell us, several other benefits can be offered using the process of fermentation. In this overview only some recent developments in the health arena will be discussed, such as natural fortification with vitamins, benefits addressing obesity and increase of anti-oxidant content.
On the basis of the successful applications in dairy products [4–6], vitamin enrichment in many other fermented food products should be possible. Using the high folate-producing Lactobacillus plantarum strain described by Hugenschmidt and co-workers [11], many novel fermented consumer products seem possible since many traditional fermented products involve the use of Lb. plantarum and this lactic acid bacterium can be found on almost any agricultural crop including animal produce. Vitamin fortification has been described for a large number of traditional fermented foods such as soy sauce [12], kimchi [13], and several others [14]. The work by Santos and coworkers, just show that this process could also be used for novel fermented consumer products, in this case fermented melon, where both the lactic acid bacteria Lactobacillus reuteri and Lb. plantarum showed much higher folate production on melon than on other natural substrates [9].
Fermentation for natural fortification
Fermentation for addressing obesity
Over the past 10 years, the process of fermentation has become recognized as a relative easy and one hundred
The developed world is facing a major threat in the overall health of its population. In many countries,
Current Opinion in Biotechnology 2013, 24:155–159
www.sciencedirect.com
Traditional biotechnology for new foods and beverages Hugenholtz 157
already more than half of the population is overweight and the number is growing at an alarming rate. These overweight people do not just lack discipline in that they eat too much and have too little exercise, but often have reached a serious state of illness (obesity) that needs medical attention. Food and beverage industries (are trying to) show their responsibility in these health issues and are putting major R&D efforts in developing consumer products that lead to reduced overall food and energy intake. One focus of this R&D is to reduce carbohydrate calories in food and beverages. Fermentation, almost by definition, will contribute to calorie reduction by converting sugars into organic acids or ethanol, although the maximum calorie reduction that can be reached in such fermentation processes cannot be more than 25%, since organic acids contain 3 kcal/g versus 4 kcal/g for sugar. Another approach of the food and beverage industry is to develop consumer products or ingredients that lead to early satiation of the consumer. The satiation factors can convey their activity at different moments in the sequence of eating and digestion, from sensing the product before consumption, to physically bulking the stomach or by release of signals at a later location in the digestive tract. One of the signals that have been shown to play a role in this satiety cascade, is organic acids such as propionic acid, butyric and acetic acid [15,16,17]. These studies indicate that short chain fatty acids regulate food intake through control of gut hormone expression such as peptide PYY and glucagon-like peptide. For acetic acid (28 mmol in a serving of vinegar) [18,19] and propionic acid (2.45% in bread) [20] actual dosages have been described that lead to satiety response in humans through delay of the gastric emptying rate and lowered blood glucose and insulin reponses. As these acids are typically produced by fermentation, satietyinducing fermented foods or beverages could be produced using a propionic, acetic and/or butyric acid bacterium as a starter. In South-East Asia, and especially Japan, products from acetic acid fermentation, such as vinegar, have a traditional healthy image, and several other health benefits, such as protection against inflammatory bowel diseases and colon cancer, seem to be associated with the fermentative production of short chain fatty acids [21,22].
Fermentation for increasing anti-oxidant activity Many fruits and vegetables are more-and-more appreciated for their natural content of valuable anti-oxidants, such as lycopene (tomatoes), equol (soy), resveratrol (red grapes, berries, pomegranate), hesperidin and naringin (oranges), and quercetin (fruits, vegetables). Besides protecting the fruit or fruit juice directly from oxidation and undesirable browning and flavor changes, these antioxidants are also, sometimes, claimed to convey antioxidant activity to the consumer and protect the human cells from destruction by oxidative radicals. The efficacy www.sciencedirect.com
of these anti-oxidants is mostly determined by how they are taken-up by the human body. Recent studies have shown that enzymatic and microbial activity (fermentation) can lead to conversions of several polyphenolic antioxidants such as hesperidin [23,24] and equol [25,26], leading to more efficient uptake and thus higher bioavailability of the plant polyphenols. As such, fermentation could also contribute to improved health of the consumer on the level of increased anti-oxidants and there are clinical studies indicating that this is indeed the case for the examples of orange (hesperidin) and soy (equol) [24,26]. For a more detailed overview on polyphenols in foods and their supposed health aspects, the reader is referred to another chapter in this Food Biotechnology issue [27].
Fermentation for even longer shelf-lives Traditionally fermented consumer products have relatively long shelf-lives ranging from about 4 weeks for liquid fermented dairy products to a year or longer for some dry fermented sausages and alcoholic beverages. Still, there is always a need for longer and more complete protection. The fermented foods and the fermented, nonalcoholic, beverages usually still contain large residual levels of sugar and of various amino acids, vitamins and minerals allowing growth of a large number of acidtolerant microorganisms which are mainly yeast or fungi. To address this problem, food microbiology research has devoted much attention to finding microorganisms, and especially lactic acid bacteria, that produce antifungal compounds. A number of antifungal-producers have been identified [28–30] and also some unique antifungal components have been elucidated [31,32] such as various cyclic peptides and metabolites such as 3-phenyllactic acid. A recent study by Crowley and co-workers show that such antifungal-producers can also be used for longer shelf-lives of fresh fruits such as pear and grapes [33]. When these antifungal-producers were applied by us in various fruit juices, the (fermented) products, clearly, showed longer protection against fungal growth (Figure 2). The challenge for the food industry is, now, to make sure that these antifungal fermentations still have the right flavor attributes, that they combine well with the fermentations that deliver other benefits and that the right strategy is chosen with respect to labeling legislation (f.i. in situ fermentation for flavor and other — antimicrobial — benefits will lead to other labeling than addition of fermented ingredients with the direct purpose of controlling fungal growth).
Conclusions and perspectives Fermentation can deliver many benefits to foods and beverages. Besides unique flavors and textures as exemplified in traditional fermented products such as yoghurt, cheese, soy sauce and kimchi, many novel benefits, especially with respect to health, can be conveyed via fermentation technology. All this can be achieved via Current Opinion in Biotechnology 2013, 24:155–159
158 Food biotechnology
References and recommended reading
Figure 2
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
(a)
(b)
(c)
Dobson A, O’Sullivan O, Cotter PD, Ross P, Hill C: Highthroughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain. FEMS Microbiol Lett 2011, 320:56-62.
2.
Guzel-Seydim ZB, Kok-Tas T, Greene AK, Seydim AC: Review: functional properties of kefir. Crit Rev Food Sci Nutr 2011, 51:261-268.
3.
Dufresne C, Farnworth E: Tea, kombucha and health: a review. Food Res Int 2000, 33:409-421.
4.
Burgess C, O‘connell-Motherway M, Sybesma W, Hugenholtz J, van Sinderen D: Riboflavin production in Lactococcus lactis: potential for in situ production of vitamin-enriched foods. Appl Environ Microbiol 2004, 70:5769-5777.
5.
Sybesma W, Starrenburg M, Tijsseling L, Hoefnagel MH, Hugenholtz J: Effects of cultivation conditions on folate production by lactic acid bacteria. Appl Environ Microbiol 2003, 69:4542-4548.
6.
Taranto MP, Vera JL, Hugenholtz J, De Valdez GF, Sesma F: Lactobacillus reuteri CRL1098 produces cobalamin. J Bacteriol 2003, 185:5643-5647.
7.
Tani Y: Microbial process of menaquinone production. J Nutr Sci Vitaminol 1992:251-254.
8.
Sybesma W, Burgess C, Starrenburg MJC, van Sinderen D, Hugenholtz J: Multivitamin production in Lactococcus lactis using metabolic engineering. Metabol Eng 2008, 6:109-115.
9.
Santos F, Wegkamp A, de Vos WM, Smid EJ, Hugenholtz J: Highlevel folate production in fermented foods by the B12 producer Lactobacillus reuteri JCM1112. Appl Environ Microbiol 2008, 74:3291-3294.
(d)
Current Opinion in Biotechnology
Effect of lactic acid bacterial fermentation on fungal growth in pear juice. The pear juice fermentations with Weillonella confuse strain 7 (a), Lactobacillus plantarum strain 62 (b) and Lactobacillus plantarum strain 16 (c) were conducted at 308C for 48 hours before the, forced, fungal infection was applied. A spontaneous fungal-infected pear juice was used for infection. A non-fermented pear juice (d) was also infected with the same fungi, as a control Subsequently, incubation was continued for 3 more days. The fermentation with strain Lp16 shows clear anti-fungal protection. The three lactic acid bacteria were selected from the study of Crowley et al. [33].
complete sustainable and 100% natural processes either by up-scaling existing, traditional kitchen-style processes or by using successful fermentation processes/microorganisms on novel substrates. With the use of novel genomic technologies more detailed insight has been given in some traditional fermentations such as sourdough [34], kefir [1], kimchi [35,36], cacao [37], yoghurt [38] and sausage [39] fermentations. Also several lesser-known fermented products have been characterized using these genomics tools [40,41]. It has clarified for instance which microorganisms are most active in these fermentations and how they are adapted to their specific environments. This type of experimental data is crucial when considering to up-scale these processes or when applying these microorganisms on other substrates. Such data will also be used to build comprehensive models of each fermentation process with the ultimate goal to accurately predict the outcome of (novel) fermentations. The struggles and challenges faced by the modelers are discussed in another chapter of this special Food Biotechnology issue [42]. However, some early successes of the Systems Biology approach are also reported [43,44,45] providing possible solutions for process improvement and also providing the essential insight for much more extensive use of the fermentation technology in the food and beverage industry. Current Opinion in Biotechnology 2013, 24:155–159
1.
10. LeBlanc JG, Milani C, Savoy de Giori G, Sesma F, van Sinderen D, Ventura M: Bacteria as vitamin suppliers to their host: a gut microbiota perspective. Curr Opin Biotechnol 2013, 24:160-168. 11. Hugenschmidt S, Miescher Schwenninger S, Lacroix C: Concurrent high production of natural folate and vitamin B12 using a co-culture process with Lactobacillus plantarum SM39 and Propionibacterium freudenreichii DF13. Process Biochem 2011, 46:1063-1070. A research article showing elegantly how fermentation can be used for natural fortification with both vitamin B12 and folate. 12. Champagne CP, Tompkins TA, Buckley ND, Green-Johnson JM: Effect of fermentation by pure and mixed cultures of Streptococcus thermophilus and Lactobacillus helveticus on isoflavone and B-vitamin content of a fermented soy beverage. Food Microbiol 2010, 27:968-972. 13. Kwak CS, Lee MS, Lee HJ, Whang JY, Park SC: Dietary source of vitamin B12 intake and vitamin B12 status in female elderly Korean s aged 85 years and older living in rural area. Nutr Res Pract 2010, 4:229-234. 14. LeBlanc JG, Laino JE, del Valle MJ, Vannini V, van Sinderen D, Taranto MP, de Valdez GF, de Giori GS, Sesma F: B-group vitamin production by lactic acid bacteria — current knowledge and potential applications. J Appl Microbiol 2011, 111:1297-1309. 15. Arora T, Sharma R, Frost G: Propionate. Anti-obesity and satiety enhancing factor? Appetite 2011, 56:511-515. 16. Hua VL, Andrea F., Edward JK Jr, Andrea RN, Mofei ML, Jennifer RK, James AH, Szeto D, Yao X, Forrest G, Marsh DJ: Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3independent mechanisms. PLoS ONE 2012, 7:e35240. A research article showing how butyrate and propionate intake can protect against diet-induced obesity. www.sciencedirect.com
Traditional biotechnology for new foods and beverages Hugenholtz 159
17. Hamer HM, Jonkers D, Venema K, Vanhoutvin S, Troost FJ, Brummer RJ: Review article: the role of butyrate on colonic function. Aliment Pharmocol Ther 2008, 27:104-119.
33. Crowley S, Mahony J, van Sinderen D: Broad-spectrum antifungal-producing lactic acid bacteria and their application in fruit models. Folia Microbiol 2012. Epubl 17 Nov 2012.
18. Mettler S, Schwarz I, Colombiani PC: Additive postprandial blood glucose-attenuating and satiety-enhancing effect of cinnamon and acetic acid. Nutr Res 2009, 29:723-727.
34. Vogel RF, Pavlovic M, Ehrmann MA, Wiezer A, Liesegang H, Offschanka S, Voget S, Angelov A, Bo¨cker G, Liebl W: Genomic analysis reveals Lactobacillus sanfranciscensis as stable element in traditional sourdoughs. Microb Cell Fact 2011, 10(suppl 1):S6.
19. Ostman E, Granfeldt Y, Persson L, Bjo¨rk I: Vinegar supplementation lowers glucose and insulin responses and increases satiety after a bread meal in healthy subjects. Eur J Clin Nutr 2005, 59:983-988. 20. Darwiche G, Ostman EM, Liljeberg HG, Kallinen N, Bjo¨rgell O, Bjo¨rck IM, Almer LO: Measurements of the gastric emptying rate by use of ultrasonography: studies in humans using bread with added sodium propionate. Am J Clin Nutr 2001, 74:254-258. 21. D’Argenio G, Mazzacca G: Short-chain fatty acid in the human colon. Relation to inflammatory bowel diseases and colon cancer. Adv Exp Med Biol 1999, 472:149-158. 22. Wong JM, de Souza R, Kendall CW, Emam A, Jenkins DJ: Colonic health: fermentation and short chain fatty acids. J Clin Gastroenterol 2006, 40:235-243. 23. Jin MJ, Kim U, Kim IS, Kim Y, Han SB, Kim DH, Kwon OS, Yoo HH: Effects of gut microflora on pharmacokinetics of hesperidin: a study on non-antibiotic and pseudo-germ-free rats. J Toxicol Environ Health A 2010, 73:1441-1450. 24. Nielsen IL, Chee WS, Poulsen L, Offord-Cavin E, Rasmussen SE, Frederiksen H, Enslen M, Barron D, Horcadaja MN, Williamson G: Bioavailability is improved by enzymatic modification of the citrus flavonoid hesperidin in humans: a randomized, doubleblind, crossover trial. J Nutrition 2006, 136:404-408. 25. Di Cagno R, Mazzacane F, Rizello CG, Vincentini O, Silano M, Guilani G, De Angelis M, Gobetti M: Synthesis of isoflavone aglycones and equol in soy milks fermented by food-related lactic acid bacteria and their effect on human intestinal Caco2 cells. J Agric Food Chem 2010, 58:10338-10446. 26. Chien HL, Huang HY, Chou CC: Transformation of isoflavone phytoestrogens during the fermentation of soymilk with lactic acid bacteria and bifidobacteria. Food Microbiol 2006, 23:772-778. 27. Bolca S, Van de Wiele T, Possemiers S: Gut metabotypes govern health effects of dietary polyphenols. Curr Opin Biotechnol 2013, 24:220-225. 28. Miescher Schwenninger S, Meile L, Lacroix C: Antifungal lactic acid bacteria and propionibacteria for food biopreservation. In: Protective cultures, antimicrobial metabolites and bacteriophages for food and beverage biopreservation. Woodhead Publ Ser Food Sci Technol Nutr 2011, 201:27-62. 29. Dalie´ DKD, Deschamps AM, Richard-Forget F: Lactic acid bacteria – potential for control of mould growth and mycotoxins: a review. Food Control 2010, 21:370-380. 30. Tu˚ma S, Vogensen FK, Plockova´ M, Chumchalova´ J: Isolation of antifungally active lactobacilli from Edam cheese. Acta Alimentaria 2007, 36:405-414. 31. Miescher Schwenninger S, Lacroix C, Truttmann S, Jans C, Bommer D, Spo¨rndli C, Bigler L, Meile L: Characterization of lowmolecular weight antiyeast metabolites produced by a foodprotective Lactobacillus/Propionibacterium co-culture. J Food Prot 2008, 71:2481-2487. 32. Stro¨m K, Sjo¨gren J, Broberg A, Schnu¨rer J: Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides Cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3phenyllactic acid. Appl Environ Microbiol 2002, 68:4322-4327.
www.sciencedirect.com
35. Jung JY, Lee SH, Kim JM, Park MS, Bae J-W, Hahn Y, Madsen EL, Jeon CO: Metagenomic analysis of kimchi, a traditional Korean fermented food. Appl Environ Microbiol 2011, 77:2264-2274. 36. Nam Y-D, Chang H-W, Kim K-H, Roh SW, Bae J-W: Metatranscriptome analysis of lactic acid bacteria during kimchi fermentation with genome-probing microarrays. Int J Food Microbiol 2009, 130:140-146. 37. Illeghems K, De Vuyst L, Papalexandratou Z, Weckx S: Phylogenetic analysis of a spontaneous cocoa bean fermentation metagenome reveals new insights into its bacterial and fungal community diversity. PLoS ONE 2012, 7:e38040. A nice example of how metagenomics can be used to understand a traditional fermentation process. 38. Sieuwerts S, Molenaar D, van Hijum SAFT, Beerthuyzen M, Stevens MJA, Janssen PWM, Ingham CJ, de Bok FAM, de Vos WM, van Hylckama Vlieg JET: Mixed-culture transcriptome analysis reveals the molecular basis of mixed-culture growth in Streptococcus thermophilus and Lactobacillus bulgaricus. Appl Environ Microbiol 2010, 76:7775-7784. 39. Nyquist OL, McLeod A, Brede DA, Snipen L, Aakra A˚, Nes IF: Comparative genomics of Lactobacillus sakei with emphasis on strains from meat. Molecular genetics and genomics. MGG 2011, 285:297-311. 40. Sakamoto N, Tanaka S, Sonomoto K: Nakayama J: 16S rRNA pyrosequencing-based investigation of the bacterial community in nukadoko, a pickling bed of fermented rice bran. Int J Food Microbiol 2011, 144:352-359. 41. Jung M-J, Nam Y-D, Roh SW, Bae J-W: Unexpected convergence of fungal and bacterial communities during fermentation of traditional Korean alcoholic beverages inoculated with various natural starters. Food Microbiol 2012, 30:112-123. A very nice example of how genomics is used for discovery of novel interactions within mixed fermentations. 42. dos Santos FB, de Vos WM, Teusink B: Towards metagenomescale models for industrial applications–the case of Lactic Acid Bacteria. Curr Opin Biotechnol 2013, 24:200-206. 43. Goffin P, van De Bunt B, Giovane M, Leveau JHJ, Ho¨ppener Ogawa S, Teusink B, Hugenholtz J: Understanding the physiology of Lactobacillus plantarum at zero growth. Mol Syst Biol 2010, 6:9. A genomics and modeling study showing evidence for plant–microbe interaction in Lb plantarum. 44. Pastink MI, Teusink B, Hols P, Visser S, de Vos WM, Hugenholtz J: Genome-scale model of Streptococcus thermophilus LMG18311 for metabolic comparison of lactic acid bacteria. Appl Environ Microbiol 2009, 75:3627-3633. 45. Teusink B, Wiersma A, Molenaar D, Francke C, de Vos WM, Siezen RJ, Smid EJ: Analysis of growth of Lactobacillus plantarum WCFS1 on a complex medium using a genome-scale metabolic model. J Biol Chem 2006, 281:40041-40048.
Current Opinion in Biotechnology 2013, 24:155–159
Traditional biotechnology for new foods and beverages Jeroen Hugenholtz The food and beverage industry is re-discovering fermentation as a crucial step in product innovation. Fermentation can provide various benefits such as unique flavor, health and nutrition, texture and safety (shelf life), while maintaining a 100% natural label. In this review several examples are presented on how fermentation is used to replace, modify or improve current, artificially produced, foods and beverages and how also fermentation can be used for completely novel consumer products. Address Swammerdam Institute for Life Sciences, University of Amsterdam and Coca-Cola Corporate Research, Mainburger Strasse 19, 84072 Au/ Hallertau, Germany Corresponding author: Hugenholtz, Jeroen ([email protected])
Current Opinion in Biotechnology 2013, 24:155–159 This review comes from a themed issue on Food biotechnology Edited by Elaine E Vaughan and Jeroen Hugenholtz For a complete overview see the Issue and the Editorial Available online 8th February 2013 0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.01.001
Introduction The process of fermentation was traditionally leveraged by the human population to increase the shelf life of perishable agricultural produce such as milk, vegetables and meat. This has resulted in a vast variety of fermented foods and beverages that is still the main part of the human diet in many under-developed countries and in most countries in South-East Asia. In North America and Europe which have extremely efficient and rapid distribution systems and an overall availability of cooling and freezing systems, most of the traditional fermented products, with the exception of fermented dairy (yoghurt and cheese) and meat (sausages) have been replaced by fresh agricultural produce, making the process of fermentation obsolete. This trend away from fermentation seems to have come to a recent stop and is now gradually reversing. More and more people (and food companies) are regaining interest in traditional and more natural foods and there is a growing dislike for the processing and energyinput that is needed to maintain freshness of agricultural crops. In addition, the food and beverage industry is continuously trying to innovate within the constraints of sustainability and naturalness. All these recent develwww.sciencedirect.com
opments are leading to increased interest and activity in fermentation technology by all consumer goods industries, big and small. This trend is not really visible in the scientific literature and (not yet) in the patent literature, so some referencing will be made to company-websites as support of statements made in this contribution.
Fermentation for all-natural foods or beverages Fermentation, nowadays, is all about bringing unique signature flavors and other benefits to consumer products in a 100% natural way. This is done using, mainly, two different approaches. The ‘easiest’ is to turn a traditional, home-grown, product into a large scale process. Examples are the dairy product, kefir [1], which is a traditional liquid fermented dairy product using a mixture of lactic acid bacteria, yeast and fungi for the fermentation of milk, resulting in a yoghurt-like, slightly alcoholic product which has traditionally been consumed by millions of people in, especially, Eastern Europe and supposedly conveys spectacular health benefits to the consumer [2]. The fermentation process is, typically, conducted at home on, or close to, the stove and using small leftovers of previously produced kefir or a small kefir granule purchased at the local grocery store, as inoculum. Only recently, the larger dairy companies have managed to upscale this process to an industrial scale using active and stable starter cultures just as is the daily practice for mainstream dairy products such as yoghurt and cheese. Another example is the traditional drink of Kvass (http:// rbth.ru/articles/2012/06/22/kvass_russias_real_national_drink_15952.html), which is a result of fermentation of kitchen left-overs mainly consisting of bread bits. This fermented product is also traditionally made at home with a final composition depending on the type of left-overs used and the nature of the locally evolved microbial culture. This traditional beverage is now also marketed by larger companies, also outside the originating country, Russia. The main challenge of these companies is to prepare this family drink with a constant quality and, especially, without alcohol. This is basically done by using controlled fermentations with standardized starter cultures and similar fermentation conditions (short, low temperature) as used for non-alcoholic beer production. Several more examples can be found of food and beverage companies launching new or improved fermented products based on traditional biotechnology [3] (http://bycristinaybarra.com/2012/10/27/burnarj-the-firstsparkling-drink-with-andalusian-taste/, http://www.calpis. net/features/story/index.html, www.the-spirit-of-georgia. de, http://www.doehler.com/en/our_products/beverage_ and_dairy_bases/fermented_products) (Figure 1). Current Opinion in Biotechnology 2013, 24:155–159
156 Food biotechnology
Figure 1
Kefiy
Boza
Amazake
Amasi
Fruit vinegars
Tr fe adi r pr me tion o d n t al uc ed ts
Kombucha
Lassi
Kvass
Current Opinion in Biotechnology
Example of traditional fermented beverages from across the world.
A second approach followed by many consumer goods companies is to transfer a successful, large-scale, fermentation process to other substrates. One typical example is, again, kefir, which traditionally is a dairy product, using milk as substrate. The newest development is to use the traditional kefir culture for fermentation of other substrates, such as fruit juice, resulting in products called ‘fruit kefir’ or something similar. Other examples are biological soft drinks such as Bionade (http://dizzyfrinks. com/drink/bionade-holunder-elderberry/) (in Germany, Switzerland, Austria) and Tumult (http://news.softpedia. com/news/Coca-Cola-Comes-Out-with-Tumult-the-SoftDrink-that-Tastes-like-Beer-216150.shtml) (in France) where the microorganisms of the traditional, fermented tea drink Kombucha [3] have been used for fermentation of malt, leading to a non-alcoholic soft drink with unique flavor for the adult market.
percent natural technology to raise vitamin levels in foods and beverages. Numerous examples, especially involving dairy applications, have been presented showing natural enrichment with riboflavin [4], folate [5], vitamin B12 [6], vitamin K2 [7] and sometimes several of these vitamins, simultaneously [8,9]. The latest developments in this area are discussed in another chapter of this Special issue [10].
In addition to offering unique sensory sensations, as the label on the package or the advertisements will most likely tell us, several other benefits can be offered using the process of fermentation. In this overview only some recent developments in the health arena will be discussed, such as natural fortification with vitamins, benefits addressing obesity and increase of anti-oxidant content.
On the basis of the successful applications in dairy products [4–6], vitamin enrichment in many other fermented food products should be possible. Using the high folate-producing Lactobacillus plantarum strain described by Hugenschmidt and co-workers [11], many novel fermented consumer products seem possible since many traditional fermented products involve the use of Lb. plantarum and this lactic acid bacterium can be found on almost any agricultural crop including animal produce. Vitamin fortification has been described for a large number of traditional fermented foods such as soy sauce [12], kimchi [13], and several others [14]. The work by Santos and coworkers, just show that this process could also be used for novel fermented consumer products, in this case fermented melon, where both the lactic acid bacteria Lactobacillus reuteri and Lb. plantarum showed much higher folate production on melon than on other natural substrates [9].
Fermentation for natural fortification
Fermentation for addressing obesity
Over the past 10 years, the process of fermentation has become recognized as a relative easy and one hundred
The developed world is facing a major threat in the overall health of its population. In many countries,
Current Opinion in Biotechnology 2013, 24:155–159
www.sciencedirect.com
Traditional biotechnology for new foods and beverages Hugenholtz 157
already more than half of the population is overweight and the number is growing at an alarming rate. These overweight people do not just lack discipline in that they eat too much and have too little exercise, but often have reached a serious state of illness (obesity) that needs medical attention. Food and beverage industries (are trying to) show their responsibility in these health issues and are putting major R&D efforts in developing consumer products that lead to reduced overall food and energy intake. One focus of this R&D is to reduce carbohydrate calories in food and beverages. Fermentation, almost by definition, will contribute to calorie reduction by converting sugars into organic acids or ethanol, although the maximum calorie reduction that can be reached in such fermentation processes cannot be more than 25%, since organic acids contain 3 kcal/g versus 4 kcal/g for sugar. Another approach of the food and beverage industry is to develop consumer products or ingredients that lead to early satiation of the consumer. The satiation factors can convey their activity at different moments in the sequence of eating and digestion, from sensing the product before consumption, to physically bulking the stomach or by release of signals at a later location in the digestive tract. One of the signals that have been shown to play a role in this satiety cascade, is organic acids such as propionic acid, butyric and acetic acid [15,16,17]. These studies indicate that short chain fatty acids regulate food intake through control of gut hormone expression such as peptide PYY and glucagon-like peptide. For acetic acid (28 mmol in a serving of vinegar) [18,19] and propionic acid (2.45% in bread) [20] actual dosages have been described that lead to satiety response in humans through delay of the gastric emptying rate and lowered blood glucose and insulin reponses. As these acids are typically produced by fermentation, satietyinducing fermented foods or beverages could be produced using a propionic, acetic and/or butyric acid bacterium as a starter. In South-East Asia, and especially Japan, products from acetic acid fermentation, such as vinegar, have a traditional healthy image, and several other health benefits, such as protection against inflammatory bowel diseases and colon cancer, seem to be associated with the fermentative production of short chain fatty acids [21,22].
Fermentation for increasing anti-oxidant activity Many fruits and vegetables are more-and-more appreciated for their natural content of valuable anti-oxidants, such as lycopene (tomatoes), equol (soy), resveratrol (red grapes, berries, pomegranate), hesperidin and naringin (oranges), and quercetin (fruits, vegetables). Besides protecting the fruit or fruit juice directly from oxidation and undesirable browning and flavor changes, these antioxidants are also, sometimes, claimed to convey antioxidant activity to the consumer and protect the human cells from destruction by oxidative radicals. The efficacy www.sciencedirect.com
of these anti-oxidants is mostly determined by how they are taken-up by the human body. Recent studies have shown that enzymatic and microbial activity (fermentation) can lead to conversions of several polyphenolic antioxidants such as hesperidin [23,24] and equol [25,26], leading to more efficient uptake and thus higher bioavailability of the plant polyphenols. As such, fermentation could also contribute to improved health of the consumer on the level of increased anti-oxidants and there are clinical studies indicating that this is indeed the case for the examples of orange (hesperidin) and soy (equol) [24,26]. For a more detailed overview on polyphenols in foods and their supposed health aspects, the reader is referred to another chapter in this Food Biotechnology issue [27].
Fermentation for even longer shelf-lives Traditionally fermented consumer products have relatively long shelf-lives ranging from about 4 weeks for liquid fermented dairy products to a year or longer for some dry fermented sausages and alcoholic beverages. Still, there is always a need for longer and more complete protection. The fermented foods and the fermented, nonalcoholic, beverages usually still contain large residual levels of sugar and of various amino acids, vitamins and minerals allowing growth of a large number of acidtolerant microorganisms which are mainly yeast or fungi. To address this problem, food microbiology research has devoted much attention to finding microorganisms, and especially lactic acid bacteria, that produce antifungal compounds. A number of antifungal-producers have been identified [28–30] and also some unique antifungal components have been elucidated [31,32] such as various cyclic peptides and metabolites such as 3-phenyllactic acid. A recent study by Crowley and co-workers show that such antifungal-producers can also be used for longer shelf-lives of fresh fruits such as pear and grapes [33]. When these antifungal-producers were applied by us in various fruit juices, the (fermented) products, clearly, showed longer protection against fungal growth (Figure 2). The challenge for the food industry is, now, to make sure that these antifungal fermentations still have the right flavor attributes, that they combine well with the fermentations that deliver other benefits and that the right strategy is chosen with respect to labeling legislation (f.i. in situ fermentation for flavor and other — antimicrobial — benefits will lead to other labeling than addition of fermented ingredients with the direct purpose of controlling fungal growth).
Conclusions and perspectives Fermentation can deliver many benefits to foods and beverages. Besides unique flavors and textures as exemplified in traditional fermented products such as yoghurt, cheese, soy sauce and kimchi, many novel benefits, especially with respect to health, can be conveyed via fermentation technology. All this can be achieved via Current Opinion in Biotechnology 2013, 24:155–159
158 Food biotechnology
References and recommended reading
Figure 2
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
(a)
(b)
(c)
Dobson A, O’Sullivan O, Cotter PD, Ross P, Hill C: Highthroughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain. FEMS Microbiol Lett 2011, 320:56-62.
2.
Guzel-Seydim ZB, Kok-Tas T, Greene AK, Seydim AC: Review: functional properties of kefir. Crit Rev Food Sci Nutr 2011, 51:261-268.
3.
Dufresne C, Farnworth E: Tea, kombucha and health: a review. Food Res Int 2000, 33:409-421.
4.
Burgess C, O‘connell-Motherway M, Sybesma W, Hugenholtz J, van Sinderen D: Riboflavin production in Lactococcus lactis: potential for in situ production of vitamin-enriched foods. Appl Environ Microbiol 2004, 70:5769-5777.
5.
Sybesma W, Starrenburg M, Tijsseling L, Hoefnagel MH, Hugenholtz J: Effects of cultivation conditions on folate production by lactic acid bacteria. Appl Environ Microbiol 2003, 69:4542-4548.
6.
Taranto MP, Vera JL, Hugenholtz J, De Valdez GF, Sesma F: Lactobacillus reuteri CRL1098 produces cobalamin. J Bacteriol 2003, 185:5643-5647.
7.
Tani Y: Microbial process of menaquinone production. J Nutr Sci Vitaminol 1992:251-254.
8.
Sybesma W, Burgess C, Starrenburg MJC, van Sinderen D, Hugenholtz J: Multivitamin production in Lactococcus lactis using metabolic engineering. Metabol Eng 2008, 6:109-115.
9.
Santos F, Wegkamp A, de Vos WM, Smid EJ, Hugenholtz J: Highlevel folate production in fermented foods by the B12 producer Lactobacillus reuteri JCM1112. Appl Environ Microbiol 2008, 74:3291-3294.
(d)
Current Opinion in Biotechnology
Effect of lactic acid bacterial fermentation on fungal growth in pear juice. The pear juice fermentations with Weillonella confuse strain 7 (a), Lactobacillus plantarum strain 62 (b) and Lactobacillus plantarum strain 16 (c) were conducted at 308C for 48 hours before the, forced, fungal infection was applied. A spontaneous fungal-infected pear juice was used for infection. A non-fermented pear juice (d) was also infected with the same fungi, as a control Subsequently, incubation was continued for 3 more days. The fermentation with strain Lp16 shows clear anti-fungal protection. The three lactic acid bacteria were selected from the study of Crowley et al. [33].
complete sustainable and 100% natural processes either by up-scaling existing, traditional kitchen-style processes or by using successful fermentation processes/microorganisms on novel substrates. With the use of novel genomic technologies more detailed insight has been given in some traditional fermentations such as sourdough [34], kefir [1], kimchi [35,36], cacao [37], yoghurt [38] and sausage [39] fermentations. Also several lesser-known fermented products have been characterized using these genomics tools [40,41]. It has clarified for instance which microorganisms are most active in these fermentations and how they are adapted to their specific environments. This type of experimental data is crucial when considering to up-scale these processes or when applying these microorganisms on other substrates. Such data will also be used to build comprehensive models of each fermentation process with the ultimate goal to accurately predict the outcome of (novel) fermentations. The struggles and challenges faced by the modelers are discussed in another chapter of this special Food Biotechnology issue [42]. However, some early successes of the Systems Biology approach are also reported [43,44,45] providing possible solutions for process improvement and also providing the essential insight for much more extensive use of the fermentation technology in the food and beverage industry. Current Opinion in Biotechnology 2013, 24:155–159
1.
10. LeBlanc JG, Milani C, Savoy de Giori G, Sesma F, van Sinderen D, Ventura M: Bacteria as vitamin suppliers to their host: a gut microbiota perspective. Curr Opin Biotechnol 2013, 24:160-168. 11. Hugenschmidt S, Miescher Schwenninger S, Lacroix C: Concurrent high production of natural folate and vitamin B12 using a co-culture process with Lactobacillus plantarum SM39 and Propionibacterium freudenreichii DF13. Process Biochem 2011, 46:1063-1070. A research article showing elegantly how fermentation can be used for natural fortification with both vitamin B12 and folate. 12. Champagne CP, Tompkins TA, Buckley ND, Green-Johnson JM: Effect of fermentation by pure and mixed cultures of Streptococcus thermophilus and Lactobacillus helveticus on isoflavone and B-vitamin content of a fermented soy beverage. Food Microbiol 2010, 27:968-972. 13. Kwak CS, Lee MS, Lee HJ, Whang JY, Park SC: Dietary source of vitamin B12 intake and vitamin B12 status in female elderly Korean s aged 85 years and older living in rural area. Nutr Res Pract 2010, 4:229-234. 14. LeBlanc JG, Laino JE, del Valle MJ, Vannini V, van Sinderen D, Taranto MP, de Valdez GF, de Giori GS, Sesma F: B-group vitamin production by lactic acid bacteria — current knowledge and potential applications. J Appl Microbiol 2011, 111:1297-1309. 15. Arora T, Sharma R, Frost G: Propionate. Anti-obesity and satiety enhancing factor? Appetite 2011, 56:511-515. 16. Hua VL, Andrea F., Edward JK Jr, Andrea RN, Mofei ML, Jennifer RK, James AH, Szeto D, Yao X, Forrest G, Marsh DJ: Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3independent mechanisms. PLoS ONE 2012, 7:e35240. A research article showing how butyrate and propionate intake can protect against diet-induced obesity. www.sciencedirect.com
Traditional biotechnology for new foods and beverages Hugenholtz 159
17. Hamer HM, Jonkers D, Venema K, Vanhoutvin S, Troost FJ, Brummer RJ: Review article: the role of butyrate on colonic function. Aliment Pharmocol Ther 2008, 27:104-119.
33. Crowley S, Mahony J, van Sinderen D: Broad-spectrum antifungal-producing lactic acid bacteria and their application in fruit models. Folia Microbiol 2012. Epubl 17 Nov 2012.
18. Mettler S, Schwarz I, Colombiani PC: Additive postprandial blood glucose-attenuating and satiety-enhancing effect of cinnamon and acetic acid. Nutr Res 2009, 29:723-727.
34. Vogel RF, Pavlovic M, Ehrmann MA, Wiezer A, Liesegang H, Offschanka S, Voget S, Angelov A, Bo¨cker G, Liebl W: Genomic analysis reveals Lactobacillus sanfranciscensis as stable element in traditional sourdoughs. Microb Cell Fact 2011, 10(suppl 1):S6.
19. Ostman E, Granfeldt Y, Persson L, Bjo¨rk I: Vinegar supplementation lowers glucose and insulin responses and increases satiety after a bread meal in healthy subjects. Eur J Clin Nutr 2005, 59:983-988. 20. Darwiche G, Ostman EM, Liljeberg HG, Kallinen N, Bjo¨rgell O, Bjo¨rck IM, Almer LO: Measurements of the gastric emptying rate by use of ultrasonography: studies in humans using bread with added sodium propionate. Am J Clin Nutr 2001, 74:254-258. 21. D’Argenio G, Mazzacca G: Short-chain fatty acid in the human colon. Relation to inflammatory bowel diseases and colon cancer. Adv Exp Med Biol 1999, 472:149-158. 22. Wong JM, de Souza R, Kendall CW, Emam A, Jenkins DJ: Colonic health: fermentation and short chain fatty acids. J Clin Gastroenterol 2006, 40:235-243. 23. Jin MJ, Kim U, Kim IS, Kim Y, Han SB, Kim DH, Kwon OS, Yoo HH: Effects of gut microflora on pharmacokinetics of hesperidin: a study on non-antibiotic and pseudo-germ-free rats. J Toxicol Environ Health A 2010, 73:1441-1450. 24. Nielsen IL, Chee WS, Poulsen L, Offord-Cavin E, Rasmussen SE, Frederiksen H, Enslen M, Barron D, Horcadaja MN, Williamson G: Bioavailability is improved by enzymatic modification of the citrus flavonoid hesperidin in humans: a randomized, doubleblind, crossover trial. J Nutrition 2006, 136:404-408. 25. Di Cagno R, Mazzacane F, Rizello CG, Vincentini O, Silano M, Guilani G, De Angelis M, Gobetti M: Synthesis of isoflavone aglycones and equol in soy milks fermented by food-related lactic acid bacteria and their effect on human intestinal Caco2 cells. J Agric Food Chem 2010, 58:10338-10446. 26. Chien HL, Huang HY, Chou CC: Transformation of isoflavone phytoestrogens during the fermentation of soymilk with lactic acid bacteria and bifidobacteria. Food Microbiol 2006, 23:772-778. 27. Bolca S, Van de Wiele T, Possemiers S: Gut metabotypes govern health effects of dietary polyphenols. Curr Opin Biotechnol 2013, 24:220-225. 28. Miescher Schwenninger S, Meile L, Lacroix C: Antifungal lactic acid bacteria and propionibacteria for food biopreservation. In: Protective cultures, antimicrobial metabolites and bacteriophages for food and beverage biopreservation. Woodhead Publ Ser Food Sci Technol Nutr 2011, 201:27-62. 29. Dalie´ DKD, Deschamps AM, Richard-Forget F: Lactic acid bacteria – potential for control of mould growth and mycotoxins: a review. Food Control 2010, 21:370-380. 30. Tu˚ma S, Vogensen FK, Plockova´ M, Chumchalova´ J: Isolation of antifungally active lactobacilli from Edam cheese. Acta Alimentaria 2007, 36:405-414. 31. Miescher Schwenninger S, Lacroix C, Truttmann S, Jans C, Bommer D, Spo¨rndli C, Bigler L, Meile L: Characterization of lowmolecular weight antiyeast metabolites produced by a foodprotective Lactobacillus/Propionibacterium co-culture. J Food Prot 2008, 71:2481-2487. 32. Stro¨m K, Sjo¨gren J, Broberg A, Schnu¨rer J: Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides Cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3phenyllactic acid. Appl Environ Microbiol 2002, 68:4322-4327.
www.sciencedirect.com
35. Jung JY, Lee SH, Kim JM, Park MS, Bae J-W, Hahn Y, Madsen EL, Jeon CO: Metagenomic analysis of kimchi, a traditional Korean fermented food. Appl Environ Microbiol 2011, 77:2264-2274. 36. Nam Y-D, Chang H-W, Kim K-H, Roh SW, Bae J-W: Metatranscriptome analysis of lactic acid bacteria during kimchi fermentation with genome-probing microarrays. Int J Food Microbiol 2009, 130:140-146. 37. Illeghems K, De Vuyst L, Papalexandratou Z, Weckx S: Phylogenetic analysis of a spontaneous cocoa bean fermentation metagenome reveals new insights into its bacterial and fungal community diversity. PLoS ONE 2012, 7:e38040. A nice example of how metagenomics can be used to understand a traditional fermentation process. 38. Sieuwerts S, Molenaar D, van Hijum SAFT, Beerthuyzen M, Stevens MJA, Janssen PWM, Ingham CJ, de Bok FAM, de Vos WM, van Hylckama Vlieg JET: Mixed-culture transcriptome analysis reveals the molecular basis of mixed-culture growth in Streptococcus thermophilus and Lactobacillus bulgaricus. Appl Environ Microbiol 2010, 76:7775-7784. 39. Nyquist OL, McLeod A, Brede DA, Snipen L, Aakra A˚, Nes IF: Comparative genomics of Lactobacillus sakei with emphasis on strains from meat. Molecular genetics and genomics. MGG 2011, 285:297-311. 40. Sakamoto N, Tanaka S, Sonomoto K: Nakayama J: 16S rRNA pyrosequencing-based investigation of the bacterial community in nukadoko, a pickling bed of fermented rice bran. Int J Food Microbiol 2011, 144:352-359. 41. Jung M-J, Nam Y-D, Roh SW, Bae J-W: Unexpected convergence of fungal and bacterial communities during fermentation of traditional Korean alcoholic beverages inoculated with various natural starters. Food Microbiol 2012, 30:112-123. A very nice example of how genomics is used for discovery of novel interactions within mixed fermentations. 42. dos Santos FB, de Vos WM, Teusink B: Towards metagenomescale models for industrial applications–the case of Lactic Acid Bacteria. Curr Opin Biotechnol 2013, 24:200-206. 43. Goffin P, van De Bunt B, Giovane M, Leveau JHJ, Ho¨ppener Ogawa S, Teusink B, Hugenholtz J: Understanding the physiology of Lactobacillus plantarum at zero growth. Mol Syst Biol 2010, 6:9. A genomics and modeling study showing evidence for plant–microbe interaction in Lb plantarum. 44. Pastink MI, Teusink B, Hols P, Visser S, de Vos WM, Hugenholtz J: Genome-scale model of Streptococcus thermophilus LMG18311 for metabolic comparison of lactic acid bacteria. Appl Environ Microbiol 2009, 75:3627-3633. 45. Teusink B, Wiersma A, Molenaar D, Francke C, de Vos WM, Siezen RJ, Smid EJ: Analysis of growth of Lactobacillus plantarum WCFS1 on a complex medium using a genome-scale metabolic model. J Biol Chem 2006, 281:40041-40048.
Current Opinion in Biotechnology 2013, 24:155–159