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IFST Winning Articles

2019; Wiley; Volume: 33; Issue: 3 Linguagem: Inglês

10.1002/fsat.3303_4.x

2689-1816

Microbial Metabolites in Food Biotechnology

Food Science and TechnologyVolume 33, Issue 3 p. 14-18 IFST Winning ArticlesFree Access IFST Winning Articles First published: 13 December 2019 https://doi.org/10.1002/fsat.3303_4.xAboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat YOUNG SCIENTIST, UNDERGRADUATE WINNING ARTICLE BY ALICE NIELD, FOOD SCIENCE GRADUATE, UNIVERSITY OF READING Aerobic growth of Lactobacillus plantarum for production of GOS prebiotics Your gut bacteria can weigh up to 2kg, that is more than the weight of the average human brain1. Modulation of the human gut microbiome is associated with many health benefits such as symptom alleviation of Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD) and reductions in liver LDL cholesterol levels2, 2, 4. Probiotics, live microorganisms (or ‘good bacteria’) that confer health benefits on the host and promote wellbeing, are found in products such as Yakult and kefir5. In this research project, a specific strain of Lactobacillus plantarum (Optibiotix product LPLDL, see Figure 1) was used. Previously, this had demonstrated the ability to reduce cholesterol in a small human study. Integral to the activity of the probiotics are the prebiotics: substrates that provide nutrients for beneficial microorganisms and stimulate their growth3. They can be found naturally in products such as Jerusalem artichokes. During this research, galactooligosaccharide (GOS) prebiotics were synthesised from the β-galactosidase enzyme derived from the LPLDL strain of Lactobacillus plantarum. Figure 1Open in figure viewerPowerPoint Light microscope image of Lactobacillus plantarum (LPLDL) probiotic under x 40 magnification It is frequently stated in the media and in literature that there are numerous health benefits from ingesting pre and probiotics. The administration of prebiotics in combination with probiotics is speculated to provide additional benefits, with a synergy occurring within the gut in the form of a synbiotic6. In this case, the prebiotic product (the GOS) is targeted to selectively stimulate the growth of the cholesterol reducing probiotic bacteria and improve their survival in the host Gastro-Intestinal (GI) tract, allowing the potential for diet-mediated cholesterol reduction in those people with slightly elevated cholesterol levels. There is a huge challenge in the food industry of producing synbiotics on a large scale due to the use of anaerobic technology for growth of the probiotic species, which is not always available in manufacturing environments. Therefore, challenges in scale up are currently being adresseed. Part of this research project demonstrated viability of GOS production using aerobically grown probiotics, eliminating the need for anaerobic technology. LPLDL was grown in MRS broth with lactose for 10 hours both aerobically and anaerobically. After centrifugation and dilution of biomass with sodium phosphate buffer (pH 6.8), cells were lysed mechanically by bead beating to yield intracellular β-galactosidase crude enzyme, see Figure 2. Figure 2Open in figure viewerPowerPoint Methodology for production of β-galactosidase enzyme for production of GOS This enzyme was used to catalyse the production of GOS via the transgalactosylation reaction using lactose as the substrate. GOS are prebiotic carbohydrates comprised of 2-8 saccharide units, with one of these being a terminal glucose or galactose unit. GOS selectively stimulate Bifidobacterium and Lactobacillus at the expense of harmful bacteria, such as Staphylococcus aureus, see Figure 37. Figure 3Open in figure viewerPowerPoint Illustrating the kinetically controlled synthesis reaction for GOS from lactose, with the hydrolysis reaction occurring simultaneously to yield D-galactose and D-glucose GOS were synthesised over 36 hours, using 20 different temperature, pH and lactose concentration conditions. As lactose was consumed, it was either converted to GOS or hydrolysed to glucose and galactose (its constituent monosaccharides). GOS yields peaked between 13 and 17 hours, before glucose and galactose increased. Anaerobically, GOS yields of 36% were achieved. There was a clear compromise necessary between a high enough temperature to allow for lactose solubility yet ensuring enzyme activity was maintained throughout. When grown aerobically, β-galactosidase was produced by the LPLDL strain and GOS were synthesised albeit with lower maximum yields of 15%. This is promising for the prebiotics industry, suggesting that use of facultative anaerobic probiotics that respond well to oxidative stress could be used to produce a prebiotic, when using probiotics grown in either anaerobic or aerobic conditions. Conditions could be altered to achieve higher yields or comparable yields to that of the anaerobic growth, such as varying growth time, temperature or lactose concentration to match the growth characteristics of the probiotic under aerobic conditions. In conclusion, this research project demonstrated that GOS were produced from enzymes derived from aerobic growth of L. plantarum, offering significant improvements to current challenges in the food industry relating to anaerobic growth. This would allow for the scale up of this synbiotic technology to yield a viable product with the potential for selectively increasing probiotics with known cholesterol reducing abilities, so that cholesterol could be reduced using diet-mediated strategies in those people with slightly elevated cholesterol levels. We probably all know someone with elevated cholesterol levels, or perhaps some of you reading this have been told by your doctor to reduce your cholesterol levels. Cardiovascular disease (CVD) is the 2nd largest cause of mortality in the UK and with high LDL cholesterol being a main risk factor8, the ability to scale up this technology in industry would allow for potential reductions in cholesterol before medication becomes a necessity. Whilst you are reading this article, at least one person in the UK will have passed away from CVD9. This puts into perspective the impact this disease has both in the UK and globally. The scale up of this technology using readily available aerobic technology would be a stepping stone in both improving gut microbiology composition and reducing elevated cholesterol levels within the population (see Figure 4). Figure 4Open in figure viewerPowerPoint Strategy for improving gut microbiology composition and reducing elevated cholesterol levels Figure 1Open in figure viewerPowerPoint a) Bacillus cereus when exposed to nutmeg extract for 3h and b) E.coli when treated with nutmeg extract for 3h incubation (SEM Image at 25K x magnification) With thanks to Professor Bob Rastall and Dr Vasiliki Kachrimanidou (University of Reading) for their supervision and support and to Optibiotix for their sponsorship of this research project. YOUNG SCIENTIST, POSTGRADUATE, WINNING ARTICLE BY SARAVJEET KAUR BAJWA, MANCHESTER METROPOLITAN UNIVERSITY Antimicrobial properties of bioactive compounds of Indian spices and herbs in food Food safety has increasingly become a fundamental health concern for consumers and a major challenge for food manufacturers due to outbreaks of foodborne diseases caused by pathogenic microorganisms1. In the process of combating the impact of food-borne microorganisms, there has been increased use of synthetic chemical preservatives such as nitrites, nitrates, benzoic acid as antimicrobial agents. Studies showed that nitrites and nitrates when used in processed meat and products cause increased risk of colon cancer2, 3. The utilisation of sulphites can result in allergic responses in sulphite sensitive persons. The use of synthetic phenolic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) has been reduced, due to concerns about health risks4. Moreover, consumers are increasingly suspicious of synthetic preservatives and have increased desire for ‘CLEAN LABEL’. In the last decades, many research studies have focused on finding alternate antimicrobial agents for use in food to combat both pathogenic and spoilage microorganisms. Some of the studies have looked at potential bioactive compounds from natural plant based materials that could be used in food products, which inhibit growth of microorganisms and provide food safety and preservation effects2, 5. Plant sources of natural antimicrobials A number of spices and herbs contain natural antimicrobial properties and can be used to extend the shelf life of unprocessed and processed foods by reducing microbial growth6-8. Spices and herbs are natural plant materials, that are not only used as flavouring or colouring agents in food, but as standardised extracts for medicinal purposes, such as eugenol (from clove oil), and are also used in small quantities as preservatives in food9. Application in the preservation of foodstuff is mainly through inhibition of lipid oxidation, colour loss and as retardants of microbial activity2, 5. Commonly used Indian spices and herbs are listed in Table 1. Table 1. Some commonly used Indian spices and herbs Common name Biological name Family Part used Bay leaves Laurus nobilis Lauraceae Leaves Black pepper Piper nigrum Piperaceae Fruit Cinnamon Cinnamomum verum Lauraceae Bark Cloves Syzygim aromaticum Myrtaceae Flower buds Coriander Coriandrum sativum Apiaceae Seeds Cumin Cuminum cyminum Apiaceae Seeds Mace Myristica fragrans Myristica Aril (seed covering) Nutmeg Myristica fragrans Myristica Seed Saffron Crocus sativus Iridaceae Stigmas with style Sesame seeds Sesamum indicum Pedaliceae Seeds Thyme Thymus vulgaris Lamiaceae Leaves with flowering tips Turmeric Curcuma longa Zingiberaceae Roots Bioactive components of spices and herbs Spices and herbs contain secondary metabolites – a variety of bioactive compounds such as phenolic acids, flavonoids, and terpenes, which may be present in various parts of the plants such as flowers (jasmine, rose and lavender), buds (clove), leaves (thyme, bay leaves), fruits (star anise), twigs, bark (cinnamon), seeds (coriander, cardamom), wood (sandal), roots (ginger)5, 10 imparting antimicrobial and antioxidant properties2, 5, 11. Cinnamon, cloves, oregano, thyme and rosemary are some common spices with strong antimicrobial activity. The essential oils extracted from spices and herbs have shown antimicrobial activity inhibiting foodborne pathogens such as Listeria monocytogenes, Salmonella typhi, E.coli and Bacillus cereus in food as well as providing longer shelf life1, 3. Essential oil composition depends on internal and external agents influencing the plant such as genetic structures, ecological situations and agricultural factors12. In addition, seasonal variations, developmental stages of collected plant material, methods of harvest, processing of plant material, such as extraction methods and the conditions of analysis, influence the essential oil yield and the composition of bioactive compounds12, 13. The active components including phenols, saponin, thiosulfinates, glucosinolates, alcohols, aldehydes, ketones, ethers and hydrocarbons especially in spices, for example, cinnamon, clove, garlic, mustard and onion, show antimicrobial properties which result in inhibition of Gram-positive and Gram-negative pathogens1 (Table 2). Table 2. Bioactive (phenolic) components found in commonly used spices and herbs as a major antimicrobial compounds [Adapted from 1,14] Category Class Sub-class Example of spices and herbs Polyphenols Flavonoids Flavanols (e.g. Catechin) Cinnamon Flavanones Fennel Flavones Onion, Oregano Flavonols (e.g. Quercetin) Coriander, Cumin, Black pepper, Onion Non-Flavonoids Phenolic acids Cloves Terpenes Limonene Fenugreek, Mustard Vanilloids Curcumin Turmeric, Ginger Organosulphur compound Disulfides, Thiosulfinates Garlic, Onion These bioactive components are responsible for antimicrobial action including degradation of the cell wall, disruption of the cytoplasmic membrane, leakage of cellular components, and destruction of protein2. Figure 1 shows scanned electron microscopic (SEM) images of the effect of spice extract on Bacillus cereus and E.coli. Figure 1Open in figure viewerPowerPoint Protein content and Greenhouse Gas Emissions for various protein-rich foods5, 6 Antimicrobial properties of spices and herbs Numerous studies have shown that cinnamon, which is the world's most frequently consumed spice and has been granted generally recognised as safe (GRAS) status, is rich in bioactive compounds such as cinnamaldehyde that possesses antimicrobial effects1, 14. Tiwari et al15 reported that antimicrobial efficacy of spice extracts depends upon their chemical profile and concentration of bioactive components. Studies have shown that antimicrobial effects of essential oil extracts from spices and herbs have comparable effects to synthetic additives but their applications in the food industry has been limited due to their inherent characteristics, such as strong odour, flavour, aroma and relatively high cost1. Research studies on essential oils of spices and herbs over the past few years are listed in Table 3. Table 3. Research into essential oils of plant antimicrobials (spices and herbs) over 20 years Spices and herbs Applications Effective against Reference Oregano, thyme, coriander Effective antimicrobial components in essential oils (EOs) use in food preservation, Enterobacteria, lactic acid bacteria, B. cereus, Pseudomonas spp; Bacillus cereus, Pseudomonas aeruginosa, E.coli, Listeria monocytogenes Almajano et al16 Gutierrez et al17 Cinnamon, cloves, cumin EOs showed strong antimicrobial effect, Food flavouring and preservation Staphylococcus aureus, Klebsiella pneumonia aeruginosa, E. coli; Bacillus cereus, L. monocytogenes, Pseudomonas fluorescens, Salmonella enteritidis Ceylan and Fung18 Agaoglu et al19 Wei et al9 Bay leaves, coriander, cinnamon, thyme Effective antimicrobial properties for pathogenic spoilage microorganisms Bacillus subtilis, E.coli, L. monocytogenes, Salmonella typhimurium, Staphylococcus aureus Burt5 Gutierrez et al17 Bajpal et al20 Thyme, cinnamon, clove Effective essential oil components Bacillus cereus Davidson and Naidu21 Future of antimicrobial agents in food Studies showed that the addition of spice extracts could effectively retard microbial growth, reduce lipid oxidation, maintain or improve sensory attributes and extend the shelf life of food during storage1, 5, 22. Several studies showed that spice extracts have antimicrobial and antioxidant properties1, 5. The bioactive components in spice extracts could be relevant to use as natural antimicrobial alternatives to synthetic chemical preservatives in food safety and preservation. ■ With thanks to supervisors – Dr Tristan Dew, Dr Nessar Ahmed, Dr Daniel Anang, Department of Health Professions, Manchester Metropolitan University, UK. WINNING ARTICLE IN THE SCIENCE COMMUNICATIONS COMPETITION BY CATHRINE BAUNGAARD OF LIVERPOOL JOHN MOORES UNIVERSITY Quin-what? Cultivating exotic protein-rich crops in Europe Quinoa (kinwa)! Chances are you have heard of it, maybe even enjoyed it in a salad or stew. Thanks to innovative research and technology, exotic crops like quinoa and amaranth are making their debut out in the European fields and could be finding their way on to our dinner plates within exciting new food products. A case of unsustainable protein demand There is no doubt, British people love their protein. A cooked breakfast just isn't the same without the rashers of bacon and we wouldn't want to forget about the childhood favourite - bangers and mash. For centuries animal protein has been considered the centrepiece of the Western meal. Yet, it is no secret, our growing demand for protein is causing unprecedented damage to the natural environment. For example, our food systems are a major contributor to climate change, accounting for 30% of total global greenhouse gas emissions, of which meat and meat-derived products account for the majority (Figure 1)1! Additionally, animal derived products take up more than 50% of Europe's total water consumption, talk about being thirsty2! But that isn't the end of the story. Our over-reliance on monocultures, producing a single crop at a time, in wheat, corn and even bananas is placing our supply chains at greater risk from diseases and future climatic change, a seriously dangerous situation. In addition, as the world population continues to grow, so too will the demand for protein. Therefore, addressing how we can meet these protein needs in a healthy and sustainable way, may be the biggest challenge society and the food industry face. To meat or not to meat? That is the question and with initiatives like ‘Veganuary’ becoming more mainstream, consumers are increasingly answering, no thank you. Consumers are becoming more conscious of ‘sustainable eating’, opting for more flexitarian consumption patterns and demanding more plant-based products. A prime example was the overwhelming success of Gregg's vegan sausage roll. This rapidly growing market of plant-based products represents a major opportunity to provide more and attractive plant-based options for consumers, while meeting nutritional needs and helping with the transition to more sustainable production. But how do we merge food security, biodiversity, environmental and human health together to solve the challenges we currently face? Well, before we dig deeper, we must first understand the differences between plant- and animal-based proteins from a health perspective. The protein in our food is comprised of 20 amino acids, the building blocks of protein. Nine of these amino acids are ‘essential’, which means our bodies are incapable of creating them, they come exclusively from our diets. However, plant-based proteins lack at least one or more of these nine ‘essential’ amino acids compared to animal-based proteins, rendering them ‘incomplete’. But don't worry! Combinations of plant-based protein sources in foods eaten throughout the day are enough to deliver the ‘essential’ amino acids we need. Interestingly, certain crops and seeds are termed ‘high-quality’ proteins, because they are almost equivalent to animal proteins. For example, quinoa and amaranth are considered high-quality because they contain the ‘essential’ amino acid lysine3, while grain legumes (lentils, chickpeas) have a ‘high-quantity’ of protein (about 20%) (Figure 1)4. These crops and legumes are perfect examples of protein sources that can help increase the transition towards more plant-based proteins, leaving a positive impact on our health and environment. #PROTEIN2FOOD To address the global protein challenge, PROTEIN2FOOD is developing high-quality and quantity plant-based protein products through improved and sustainable processing and production methods. Spanning the entire food value chain, the 19 international project partners are using a variety of scientific and technological approaches to further our understanding and development of innovative solutions and products. The project has four overall aims: enhancing protein production with effective breeding techniques; increasing Europe's arable land for protein production; increasing agricultural biodiversity with novel crops; developing protein-rich food products that are so attractive consumers will choose them over animal-based proteins. Here, we focus on two examples of scientific and technological research in crop production and food processing that have originated from the project, which could help the food industry solve the challenges of protein needs and their environmental implications. Crop production Currently, the main source of plant-based protein across the EU is soy; imported from countries like Brazil, it helps to exacerbate environmental issues such as deforestation7. Therefore, identifying crops for Europe that are suitable for sustainable production methods and climate change mitigation and take nutrition into consideration are becoming increasingly important. Determining and improving genetic traits that are needed for optimum protein production could help identify the most beneficial crops to produce in Europe. Through studying the Ribonucleic Acid (RNA) – a molecule that transports genetic information to create specific proteins – transcripts of seeds and legumes, PROTEIN2FOOD has been able to determine the molecular markers for protein production. Currently, PROTEIN2FOOD has transcriptome data on three quinoa genotypes, which can further be used to develop genetic markers for protein content, particularly valuable for plant breeding8. This novel use of technology has also resulted in the discovery of two quinoa varieties that have much higher protein content compared to other varieties, helping to increase total protein production8. As such, quinoa's unique characteristics makes the seed an ideal alternative to ensuring the production of high-quality and quantity protein in preparation for our changing consumer demand and climate. Additionally, researchers are using advanced X-ray technology (micro-tomography) to create 3D images of the internal structure of the same seeds and legumes to screen for qualitative traits. Through this, researchers can examine the varying densities and textures within the seeds, as they absorb X-rays differently. Although, whilst micro-tomography is well-established in fields like biology, it is relatively novel in the agriculture-food sector. However, PROTEIN2FOOD researchers have developed X-ray scanning protocols that will help bridge the gap between genetic information (genotype) and the physical characteristics (phenotype) of these protein-rich crops, assisting researchers in determining which seeds and crops have the highest protein content, helping to increase total protein production9. Identifying the most productive crop varieties will not only help the industry take advantage of the most genetically favourable properties to maximise protein yield on limited land, it will also allow us to move towards more sustainable and well managed crop production. Identifying the most productive crop varieties will not only help the industry take advantage of the most genetically favourable properties to maximise protein yield on limited land, it will also allow us to move towards more sustainable and well managed crop production. The scientific and technological research from this project can for example, help plant-breeders target their breeding programmes to create new cultivars with improved nutritional traits, while giving Europe the opportunity to increase its diversity of protein rich crops and legumes for both production and consumption. Got milk? Well… plant-based milk? With the larger-than-life Oatly campaigns plastered across the UK, it doesn't come as a surprise that the plant-based milk market has been experiencing unprecedent growth. This trend is largely driven by consumers’ concerns for health, environmental and animal welfare issues. Currently, soya milk is the only nutritionally balanced competitor to conventional cow's milk, packing an attractive 8.7 grams of protein5. Yet, this option is not the most sustainable choice, as soya has long supply chains and is unable to grow in cold climates7. On top of this, many plant-based foods have lower sensory qualities compared to animal-based foods, therefore novel plant-based products with high consumer acceptance are necessary to support the transition to more plant-based diets. This is where lentils and PROTEIN2FOOD come into the picture. Lentils are both high in protein (31%) and contain the ‘essential’ amino acids, leucine and lysine4, as opposed to almond milk5. Using lentils, PROTEIN2FOOD has developed a novel lentil milk with a protein content similar to cow's milk which compares well based on textural, organoleptic (involving the senses) and nutritional properties to other plant-based milks4. These advances demonstrate the great potential legumes (lentils) can have in providing nutrition to the diet through valuable environmentally friendly plant-based proteins. Through the collaboration with small-to-medium sized enterprises, PROTEIN2FOOD has ensured that the ingredient composition, recipes and processes can be effectively transferred to relevant industrial product lines and environments, helping to increase market opportunities for the industry to further develop new varieties of plant-based protein products. Sharing is caring: from research to industry There can be no doubt that plant-proteins are here to stay and have the power to revolutionise the food system. However, research not communicated is research not implemented. Therefore, the advances in scientific and technological research created by projects such as PROTEIN2FOOD must be shared with the food industry to lessen our food systems’ impacts on the planet, while increasing our protein production and helping us to lead healthier and more sustainable lives. PROTEIN2FOOD is an excellent example of a multidisciplinary research project that could benefit the industry in providing novel consumer accepted plant-based products and more sustainable processing techniques. In doing so, this will not only benefit the consumers, but also the planet further down the line. PROTEIN2FOOD is not only taking plant-based protein mainstream, it hopes to make plant-based proteins great again! REFERENCES 1Pagliari, D., Piccirillo, C.A., Larbi, A., Cianci, R. 2015. The interactions between innate immunity and microbiota in gastrointestinal diseases. Journal of Immunology Research, https://doi.org/10.1155/2015/898297 2Bull, M.J., Plummer, N.T. 2014. Part 1: The Human Gut Microbiome in Health and Disease. Integrative Medicine: a Clinician's Journal 13(6): 17- 22 3Gibson, G.R., Hutkins, R., Sanders, M.E., Prescott, S.L., Reimer, R.A., Salminen, S.J., Scott, K., Stanton, C., Swanson, K.S., Cani, P.D., Verbeke, K., Reid, G. 2017. 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Available at: https://www.protein2food.eu/x-ray-microtomography-scanning-protocols-for-protein-rich-foods/ Volume33, Issue3September 2019Pages 14-18 FiguresReferencesRelatedInformation