\r\n\t- Traditionally accepted topics related to global health security,
\r\n\t- The impact of human activities and climate change on “planetary health”,
\r\n\t- The impact of global demographic changes and the emergence chronic health conditions as international health security threats.
\r\n\t- A theme dedicated to the COVID-19 Pandemic,
\r\n\t- Novel considerations, including the impact of social media and more recent technological developments on international health security.
\r\n\tThe goal of this book cycle is to provide a comprehensive compendium that will be able to stand on its own as an authoritative source of information on international health security.
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Miller",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10624.jpg",keywords:"Threats, Monitoring, Food Security, Emerging Infections, Transmission, Geopolitics, Climate Change, Cyber Health Security, COVID-19, Novel Coronavirus, Pandemic, Coronavirus",numberOfDownloads:179,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"August 20th 2020",dateEndSecondStepPublish:"November 5th 2020",dateEndThirdStepPublish:"January 4th 2021",dateEndFourthStepPublish:"March 25th 2021",dateEndFifthStepPublish:"May 24th 2021",remainingDaysToSecondStep:"3 months",secondStepPassed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"An Associate Professor of Surgery at Temple University School of Medicine and a Chair of the Department of Research and Innovation, St. Luke's University Health Network. A member of multiple editorial boards and co-author of over 550 publications.",coeditorOneBiosketch:"An Associate Professor of Surgery & Integrative Medicine at Northeast Ohio Medical University and Cardiothoracic Surgeon at the Summa Health Care System. A prolific writer and presenter, with multiple books, hundreds of peer-reviewed articles, and innumerable presentations around the world.",coeditorTwoBiosketch:"A CEO of the INDUSEM Health and Medicine Collaborative, Global Executive Director. of the American College of Academic International Medicine (ACAIM) and head of the World Academic Council of Emergency Medicine.",coeditorThreeBiosketch:"A Director of Research in the Department of Emergency Medicine at Nazareth Hospital in Philadelphia, USA, and co-chief editor of the International Journal of Critical Illness and Injury Science. A recipient of numerous local, regional, and national awards.",coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"181694",title:"Dr.",name:"Stanislaw P.",middleName:null,surname:"Stawicki",slug:"stanislaw-p.-stawicki",fullName:"Stanislaw P. Stawicki",profilePictureURL:"https://mts.intechopen.com/storage/users/181694/images/system/181694.jpeg",biography:"Stanislaw P. Stawicki, MD, MBA, FACS, FAIM, is Chair of the Department of Research of Innovation, St. Luke\\'s University Health Network, Bethlehem, Pennsylvania, and Professor of Surgery at Temple University School of Medicine. Dr. Stawicki has edited numerous books and book series on the topics of clinical research, medical education, medical leadership, patient safety, health security, and various other subjects. He is a member of multiple editorial boards and has co-authored more than 650 publications. 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His academic experience over the last two decades includes consistent achievements and innovative strategies that have led to the creation of organizations, publication of landmark papers, and commendation with prestigious citations and honors for his works that have impacted academic medicine globally. He has played a defining role in founding and building internationally recognized interdisciplinary indexed journals. He is the CEO of the INDUSEM Health and Medicine Collaborative and heads the World Academic Council of Emergency Medicine (WACEM). Additionally, he serves as the Global Executive Director for the American College of Academic International Medicine (ACAIM). 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He completed residencies in Emergency Medicine and Internal Medicine at the State University of New York (SUNY) Downstate Medical Center (2010) where he served as Chief Resident for Research. He completed fellowships in Pulmonary Medicine at the University of Pittsburgh Medical Center (2013) and Critical Care Medicine at the National Institutes of Health (2014). He is active in the American College of Academic International Medicine, and is co-chief editor of the International Journal of Critical Illness and Injury Science. 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People have been eating soybeans for almost 5,000 years. Unlike most plant foods, soybeans are high in protein. Researchers are interested in both the nutritional value and the potential health benefits of soybeans. Research of the health effects of soyfoods and soybean constituents has been received significant attention to support the health improvements or health risks observed clinically or in vitro experiments. This research includes a wide range of areas, such as cancer, coronary heart disease (cardiovascular disease), osteoporosis, cognitive function (memory related), menopausal symptoms, renal function, and many others. This chapter provides up-to-date coverage on biologically active and related organic molecules isolated from soy and soy products. Their biological activities are briefly summarized. Molecules discussed in this chapter are as follows: isoflavones, phytic acid, soy lipids, soy phytoalexins, soyasaponins, lectins, hemagglutinin, soy toxins, and vitamins.
Soy products have been considered as great source of protein for many decades. Japanese, in particular, eat a soy-based diet. Japanese monks eat soy products as their main protein source. They are known to live longer and have lower rates of chronic diseases. Because soybeans contain practically no starch, soybeans are an important part of a diabetic diet. Soybeans are 1) rich in protein (vide supra), calcium, and vitamins, and 2) high in mono-saturated fatty acids. In addition, soybeans contain several biologically interesting phytochemicals as minor components, which scientists are interested in understanding their biological functions.
Many studies have shown that American and European males have a ten-fold increase in the risk of prostate cancer development as compared with East Asian countries. The observed difference prevalence of prostate cancers is considered due, in part, to difference in soy consumption. Some studies indicated that isoflavones found in soybeans contribute to the risk reduction of prostate cancer. Although there is a lack of unequivocal evidences that consuming soy as an adult may reduce the risk of breast cancer, several researches have reported that consuming soy products as a teenager may help reduce breast cancer risk as an adult. Some medical research has determined that ingredients of soybeans may help reduce the risk of colon cancer and heart diseases. The dramatic increase in soy products is due largely to the fact that the US FDA approved soy products as an official cholesterol-lowering food, along with other heart and health benefits due to the evidence that soy product intake is correlated with significant decreases in serum cholesterol, low-density lipoprotein (LDL, bad cholesterol) and triglycerides. Although a significant health benefit has been observed in people with a high soy intake (vide supra), some scientists have argued that reported health benefits are poorly supported by the available experimental evidences. It seems to be difficult to support soy product benefit by in vitro studies using individually isolated phytochemicals (a wide variety of compounds produced by plants) or crude products prepared from soy. It is important to note that all phytochemicals isolated from soybeans show an array of weak biological activities, thus, normal consumption of foods that contain these phytochemicals should not provide sufficient amounts to elicit a visible physiological response in humans in short-time clinical researches.
Remarkably, seeds of soy contain very high levels of protein, carbohydrate conjugates, fatty acids (soybean oil), amino acids, and inorganic materials (minerals). Among these soybean components, protein and fatty acid content account for about 40% and 20%, respectively. The remaining components consist of carbohydrate conjugates, inorganic constituents, and the minor components of biologically interesting small molecules (molecules highlighted below). Thus, soybeans constitute important nutritional components. Soybeans are considered to be a good substituents of protein (essential amino acids), amongst other major vegetables, for animal products. This chapter reviews secondary metabolites isolated from soy and soy products that show interesting biological activities.
Isoflavones (a subgroup of flavonoids) are known to be highly potent antioxidants (Fig.1). As stated above, the consumption of soy products has many health benefits, including protection against breast cancer, prostate cancer, menopausal symptoms, heart disease and osteoporosis. Many of the health benefits of soy are derived from its isoflavones. Isoflavones are produced via a branch of the general phenylpropanoid pathway biosynthesis (begins from phenylalanine) that produces flavonoid compounds in legumes and stored as glucosyl- and malonyl-glucose conjugates (Graham, 1991). The major isoflavones in soybean are genistein, daidzein, and glycitein, comprising about 50, 40, and 10% of total isoflavone profiles, respectively. The chemical structure of isoflavones is similar to that of the primary female sex hormone, estrogen. Because of this similarity in structure, they can interfere with the action of estrogen. Thus, isoflavones are often called “phytoestrogens”. The common biological roles of phytoestrogens are to protect plants from stress and to act as part of a plant’s defence mechanism. Some scientists postulate that phytoestrogens may have evolved to protect the plants by interfering with the reproductive ability of grazing animals. The estrogen effects of isoflavones are much less effective than estrogen; its effectiveness represents around 1/1000 of estrogen. Isoflavones can also reduce the effect of the estrogen on cells and skin layers when the hormone levels are high, reducing the risk of estrogen linked cancers. There are two other classes (lignans and coumestans) of phytoestrogens that have estrogen-like actions in the human body. Isoflavones have been reported to show estrogenic, antifungal, anti-tumor and anti-mutagenic properties (Rishi, 2002; Dorge and Sheehan, 2002; Coward et al., 1993; Miyazawa, 1999). Isoflavones remain the subject of many scientific studies, as illustrated by the more than 18,000 scientific publications.
Structures of soy isoflavones
Genistein is found in a number of plants including soybeans, lupin, fava beans, kudzu, psoralea, and coffee. Genistein is the most discussed phytoestrogenic substance, because it is very well represented in soybeans. Genistein influences several targets in living cells. Due to its structural similarity to estrogen (i.e. 17β-estradiol, Fig. 2), genistein can bind to estrogen receptors. Genistein shows much higher affinity toward estrogen receptor β (ERβ) than toward estrogen receptor α (ERα).
Structures of estrogen
Estrogen is a key regulator of growth and differentiation in a broad range of tissues, including the reproductive (genital) system, mammary gland, central nervous and skeletal systems. Estrogen is also known to be involved in breast and endometrial cancers. To date, two key conclusions can be highlighted from the significant number of studies on the specific roles of the two receptor subtypes in diverse estrogen target tissues. ERα and ERβ have different transcriptional activities in certain cell-type, which help to explain some of the major differences in their tissue-specific biological actions. Both ERs are widely expressed in different tissue types, however, there are some distinct differences in their expression patters. The ERα is found in the inner membrane of the uterus (endometrium) and breast cancer cells. On the other hand, ERβ is found in kidney, brain, bone, heart, lung, intestinal mucosa, prostate, and endothelial cells. Unwanted effects are generally mediated through ERα. Roles of ERβ have been the subjects of interest in human cancer researches. Recent studies have shown that ERβ is lost in majority of breast tumors and thus ESR2 gene, encoding ERβ, is suggested to be a possible tumor suppressor gene. Similarly, ERβ overexpression in ovarian cancer cells is suggested to exert antitumoral effects. ERβ is highly expressed in prostate cancer cells, and is the predominant estrogen receptor in the colonic epithelium. Thus, effects of estrogen in these cells or tissues are mediated by ERβ. Therefore, non-steroidal ERβ-antagonist has potential to be a clinically useful drug.
In the 1960’s, many researches regarding the physiological effects of genistein were limited to its estrogenic activity. Genistein have also been shown to possess antifungal activities (Weidernbörner, et al. 1989), antiangiogenic effects (blocking formation of new blood vessels), and may block the uncontrolled cell growth associated with cancer, most likely by inhibiting the activity of substances in the body that regulate cell division and cell growth factors. Various studies have found moderate doses of genistein to have inhibitory effects on cancers of the prostate, cervical, brain, breast, and colon. Additionally it has been shown that genistein makes some cells more sensitive to radio-therapy. Genistein has shown a protein tyrosine kinase inhibitory activity. Tyrosine kinases are implicated in almost all cell growth and proliferation signal cascades. Genistatin’s inhibition of DNA topoisomerase II also plays an important role in the cytotoxic activity of genistein.
Daidzein is also present a number of plants. Soy foods typically contain more genistein than daidzein. Structurally, daidzein lacks the 5-hroxy group of genistein (Fig. 1). Genistein and daidzein can transfer across the human placenta at environmentally relevant levels and their influence to early puberty in children is unknown. In vitro and in vivo studies have shown that daidzein stimulates the growth of estrogen-sensitive breast cancer cells. Some epidemiological evidence indicates that soy intake may be more protective when the exposure occurs prior to puberty. More research needs to be conducted on the association between breast cancer risk and daidzein specifically before conclusions can be drawn. Daidzein is metabolized in the colon by bacteria to equol and another isoflavones. Daidzein is available as a dietary supplement.
Glycitein is unique in that it is an isoflavone found in soy with a methoxy group. Methylated isoflavones have been shown to be more bioavailable and biologically stable than non-methylated isoflavones. Glycitein accounts for 5-10% of the total isoflavones in soy food products. Glycitein shows a weak estrogenic activity, comparable to that of the other soy isoflavones.
Formononetin is an O-methylated isoflavone. It is found in the family Fabaceae and Ranunculaceae (i.e. clovers, soybeans, and cohosh). Formononetin is known to be converted in the rumen (in sheep and cow) into a potent phytoestrogen, equol. Although, O-demethylase, catalysing O-demethylation, has been attributed to metabolism by gut microflora, incubation of formononetin with human liver microsomes resulted in 4’-O-demethylation to yield daidzein (Tolleson, et al. 2002) (Scheme 1).
Biochanin A is an O-methylated isoflavone. Biochanin A can be found in red clover, soy, alfalfa sprouts, peanuts, chickpea and other legumes. Biochanin A-containing supplements are derived from red clover and, in addition to biochanin A, usually contain genistein, daidzein and formononetin. In red clover, biochanin A exists as its glycoside (Fig. 3). However, the glycoside undergoes hydrolysis during extraction to form the aglycone (non-sugar component). Biochanin A has weak estrogenic activities as measured in in vivo and in vitro assays. In comparison with other isoflavones, biochanin A is expected to have possible anti-osteoporotic activity. Structurally, biochanin A would be expected to be able to scavenge reactive oxygen species and inhibit lipid peroxidation. In vitro and in vivo studies using rodents indicated that biochanin A has anticarcinogenic activity.
Biotransformation of formonetin, daidzein, and equol from ononin.
Equol has the 3S configuration and is produced by bacterial flora in the intestines as a metabolite of daidzein (Scheme 1). However, only about 30-50% of people have intestinal bacteria that produce equol. Equol is a non-steroidal estrogen that acts as an anti-androgen by blocking the hormone dihydrotestosterone. Equol has the ability to bind to ERβ. This may make equol advantageous in estrogen-related cancers, including breast cancer. Equol is unique because it not only has the ability to bind to ERβ, but also acts as an antagonist to androgen actions. Unlike anti-androgen drugs, equol does not bind to androgen receptors, but it binds directly to dihydrotestosterone. This mode of action has prompted studies to determine if men who are equol-producers may have an advantage against prostate cancer.
Isoflavones generally exist as aglycones (Fig. 1) and their glycoside forms. Isoflavone glycosides isolated from soybeans are β-glucosidated at C7-position of isoflavone core structure. Soybeans are known to contain daidzein, glycitin, 6”-acetylgenistin, 6”-acetyldaizin (Waltz, 1931; Naim, et al. 1973; Ohta, et al. 1979). Later, malonylglycosides (6"-O-malonyldaidzin, 6"-O-malonylgenistin, 6"-O-malonylglycitin) and succinylglycosides (6"-O-succinyldaidzin, 6"-O-succinylgenistin, 6"-O-succinylglycitin) are found in soybeans or soy products (Wang et al., 1994; Toda, 1999) (Fig. 3). Careful analyses of isoflavones in soybeans revealed that the malonyglycosides are the predominant isoflavones in soybeans. Mass balance of isoflavone glycosides vary depending on manufacturing process of soy products.
As describe above, biological effects of aglycones of isoflavone glycosides found in soybeans have been of great interest in food science, food technology, nutrition and dietary supplements, and disease prevention or treatment. Due to the fact that isoflavones in soybeans are conjugated almost exclusively to sugars, thus, understanding of the mechanism of intestinal absorption of isoflavones in humans is an important subject. Evidence from intestinal perfusion and in vitro cell culture studies indicates that isoflavone glycosides are poorly absorbed, yet isoflavones are bioavailable and appear in high concentrations in plasma, irrespective of whether they are ingested as aglycones or glycoside conjugates. Therefore, it was suggested that hydrolysis of the sugar moiety is an essential prerequisite for bioavailability of soy isoflavones (Setchell et al. 2002).
Structures of soy isoflavone glycosides
Recently, a lot of articles regarding the negative aspects of soy have been published. However, several controversy reports about the adverse effects are always not clear. This may be due to the lack of understanding of metabolism and bioavailability of isoflavones in soy products. Some studies concluded that the bioavailability and pharmacokinetics of isoflavones are significantly influenced by type of soy products. Examples of adverse effects of isoflavones are that genistein 1) increased the rate of proliferation of estrogen-dependent breast cancer in vitro when not co-treated with an estrogen antagonist, 2) decreased efficiency of tamoxifen and letrozole, drugs commonly used in breast cancer therapy, and 3) inhibited immune response towards cancer cells due in part to the reduction of thyroid function. In some analyses of current concerns regarding the estrogen-like effects of isoflavones in the breast cancers on the clinical trial data and recent evidence regarding estrogen therapy use in postmenopausal women, Messinia and Wood concluded that there is little clinical evidence to suggest that isoflavones will increase breast cancer risk in healthy women or worsen the prognosis of breast cancer patients. They also pointed out that the clinical trials often involved small numbers of subjects, and there is no evidence that isoflavone intake increases breast tissue density in pre- or postmenopausal women or increases breast cell proliferation in postmenopausal women with or without a history of breast cancer. The Israeli health ministry has recommended only moderate consumption of soy products because of reported adverse effects of isoflavones (vide supra). On the other hand, the British Dietary Association concluded that evidence suggesting isoflavones reduce the symptoms of menopause is inconsistent. Although more clinical researches should be performed to definitively alleviate above concerns, the existing data should provide some degree of assurance that isoflavone exposure at levels consistent with a large amount of soy product intake does not result in adverse effects on breast tissue (Messina et al, 2008).
Phytic acid [hexakisphosphate (IP6)) or phytate] is present in the brans and hulls of most grains, beans, nuts, and seeds. Rich sources of phytic acid are wheat bran and flaxseed. Phytic acid is inositol hexaphosphate, and thus it is highly charged, which provided chelative (or binding) properties. Phytic acid binds to minerals and metals. Phytate is not digestible to humans or nonruminant animals. The chelated forms of phytic acid with Zn, Ca, and Mg make them impermeable molecules through cell membranes. Phytic acid blocks the body\'s uptake of essential minerals such as magnesium, calcium, iron and especially zinc. On the other hand, phytic acid is known to be an antioxidant as well as helpful in eradication of heavy metals and other toxic cation species from the body.
Lipids are broadly defined as hydrophobic or amphiphilic molecules. Lipids include fatty acids, sterols, lipid-soluble vitamins (vitamins A, D, E and K), glycerolipids, phospholipids, glycolipids, and sphingoglycolipid. Soybeans contain 82% of triacylglycerol, 13% of phospholipids, about 1% of sterols, and 4% of unsaturated and saturated fatty acids in a total lipid extracted with chloroform-methanol (2/1). Phospholipid composition in a soybean lipid extract is phosphatidylcholine (42%), phosphatidylethanolamine (30%), phosphatidylinositol and phosphatidylserine (20%), lysophosphatidylcholine (1%), sphingomyeline (0.6%), phosphatidic acid and others, respectively (Takagi et al. 1985).
Lipids remain an important research subject because of associations between consumption of lipids and the incidence of some chronic conditions including coronary artery disease, diabetes, cancer and obesity. Dietary lipids (or fats) serve multiple purposes. The importance of antioxidant ability of unsaturated fatty acids including β-carotene in the prevention of cardiovascular disease as well as many cancers is being increasingly recognised. Although saturated fatty acids are generally considered cholesterolemic, it is now evident that the effect of some fatty acids on blood lipids and lipoproteins suggest that the major dietary fats containing in some food products (i.e. soybeans or palm oils) do not raise plasma total fatty acids and LDL cholesterol levels. In recent times, adverse health concerns from the consumption of trans fatty acids arising from hydrogenation of oils and fats have been the subject of much discussion and controversy.
Structures of representative lipids isolated from soybeans
Soybean oil is rich in polyunsaturated fatty acids, including the two essential fatty acids, linoleic and linolenic, that are not produced in the human body. Linoleic and linolenic acids aid the body\'s absorption of vital nutrients and are required for human health.
In many applications, the higher saturate oils have been replaced with partially hydrogenated vegetable oils. Partially hydrogenated oils make the oil more stable and more resistant to air oxidation. Saturated fatty acids are more difficult to digest than unsaturated fatty acids and are seldom used for food product industry applications. Nature makes most mono- and polyunsaturated fatty acids in the cis form. However, during the partial hydrogenation process, the cis geometry of unsaturated fatty acids is partially isomerized to the trans form. Numerous research and epidemiological studies have been conducted to determine the impact of trans-fatty acids on cholesterol levels and coronary heart disease. The study by Troisi, et al. suggested a correlation between increased consumption of trans fatty acids and an increase in LDL (bad) cholesterol, which increases lipoprotein level and is an independent risk factor for the development of coronary heart disease, and decrease in HDL (good) cholesterol. This could represent an increased risk of heart attacks by trans-fatty acid intake.
Structures of plant sterols from soybeans
Soybeans contain plant sterols, β-sitosterol, β-sitostarol, campesterol, campestanol, brassicasterol, stigmasterol, and Δ5-avenasterol, and cholesterol (Fig. 6). Plant sterols are natural dietary components, and known to have serum cholesterol-lowering properties. The lowing of serum cholesterol by plant sterols is believed to be the result of an inhibition of cholesterol absorption in small intestine. Several studies suggested that unsaturated or saturated plant sterols showed different effects on cholesterol absorption and sterol excretion (Normén et al. 2007).
Glycerolipids are composed of mono-, di- and tri-substituted glycerols. In these compounds, the three hydroxy groups of glycerol are esterified (triacylglycerols) or one of hydroxy group forms the ether linkage. Subclasses of glycerolipids are represented by glycosyl glycerols and glycerophospholipids. Glycerophospholipids are subdivided into distinct classes which are characterized by the presence of one or more sugar or phosphate residues (i.e. phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidyl serine).
Soybeans contain a wide variety of triacylglyceroles, however LC-MS analyses revealed that fatty acids incorporated in soy triacylglycerols are stearic acid, palmitic acid, oleic acid, linoleic acid, and linolenic acid (Neff et al. 1995). Fatty acids in soybeans are considered to be stored as triacylglycerides.
Lecithin can easily be extracted from soybeans (or egg yolk) and soy lecithin is an additive found in many everyday foods (Fig. 5). It has low solubility in water. Due to its amphipathic characteristic, lecithin phospholipids can form either liposomes, bilayer sheets, micelles, or lamellar structures in aqueous solution. In cooking, it is sometimes used as an emulsifier and to prevent sticking (for example, in non-stick cooking spray) and or as a stabilizer in various food applications. Lecithin has been a popular supplement because it’s high choline (N,N,N-trimethylethanol) content. Choline is an essential nutrient that has benefit for heart health and brain development, as choline deficiency plays a role in liver disease, atherosclerosis, and possibly neurological disorders. It is particularly important for pregnant women to get enough choline, since low choline intake may raise the rate of neural tube defects in infants, and may affect their child\'s memory.
Phosphatidylinositol (PI) is classified as a glycerophospholipid that contains a glycerol backbone, two non-polar fatty acid tails, a phosphate group substituted with an inositol (myo-D-inositol in animals) polar head group. The most common fatty acids of PIs are stearic acid in the SN1 position and arachidonic acid in the SN2 position. Phosphatidylinositols play important roles in lipid signaling, cell signaling and membrane trafficking. The inositol ring can be phosphorylated by a variety of kinases.
Sphingolipids are structural components of eukaryotic cell membranes. A large number of recent reports have indicated that sphingolipid are involved in a number of important regulatory processes in cell development. Cerebrosides (monoglycosylceramide) is the common name for a group of glycosphingolipids.
Soya-cerebroside (Fig. 5) is a glucosylceramide isolated from soybeans, exhibited a Ca2+-binding activity. The basic structure of soya-cerebroside II including the absolute stereochemistries of (2R)-hydroxy fatty acids are identical to one of the neural glucosylceramide. However, the main long-chain base (sphingosine moiety) is C18-4,8-diunsaturated (E/Z). Biological functions of the cerebrosides in soybeans have not been thoroughly studied. Recently, a soya-cerebroside was reported to exhibit moderate tyrosinase inhibitory activity, and applied for making skin-care cosmetics for removal of (black) freckles.
Sphingomyelin is a type of sphingolipid found in animal cell membranes, especially in the membranous myelin sheath that surrounds some nerve cell axons. It consists of phosphorylcholine and ceramide (Fig.5). In humans, sphingomyelin represents ~85% of all sphingolipids. On the other hand, only 0.6% of sphingomyelin was found in a total phospholipid isolated from soybeans (Takagi, et al. 1985). The accumulation of sphingomyelin (i.e. Niemann-Pick Disease) in brain causes irreversible neurological damage. Sphingomyelin in food products is not bioavailable, and thus the accumulation of sphingomyelin in human body is not considered possible by sphingomyelin containing food intake.
Vitamin K is a lipid-soluble essential vitamin that is stable to air but susceptible to air under sunlight. The "K" is derived from the German word “koagulation”. Natural forms of vitamin K, vitamin K1 (phylloquinone) and vitamin K2 (menaquinone), exist in the human liver and other tissues at very low concentrations; vitamin K1 concentrates in the liver while vitamin K2 is well distributed to other tissues (Fig.7). Vitamin K1 is derived from dietary intake and vitamin K2 is produced by intestinal bacteria. Thus, vitamin K is not listed among the essential vitamins. Human get most of our dietary vitamin K in the form of phylloquinone (biosynthesized by plants). In prokaryotes, especially in Gram-positive bacteria, vitamin K2 will transfer two electrons in a process of aerobic or anaerobic respiration (electron transport systems). Respiration occurs in the cell membrane of prokaryotic cells. Electron donors will,with the help of another enzyme, transfer two electrons to vitamin K2. Vitamin K2, with the help of another enzyme, will in turn transfer these two electrons to an electron acceptor.
Structures of phylloquinone and menaquinone
One tablespoon of soybean oil contains about 25 μg of vitamin K1 (about 47 μg of vitamin K1 in 100g of soybeans). Green leafy vegetables and some vegetable oils are major contributors of dietary vitamin K. Nattō, a fermented Japanese soybean product, contains large amounts (approximately 870 μg per 100 grams of nattō) of vitamin K2. Vitamin K2 is known to be more effective than vitamin K1 with respect to osteroclastogenesis, hypocholesterolemic effects, and ability to slow atherosclerotic progression. To date, no adverse effects have been reported for higher levels of vitamin K intake from food and/or supplements, there are no documented toxicity symptoms for vitamin K.
In the human diet, carotenoids have been shown to have antioxidant activity which may help to prevent certain kinds of cancers, arthritis and atherosclerosis. β-Carotene is a precursor of vitamin A (retinal) which is biosynthesized via the action of β-carotene 15,15\'-monooxygenase. There are nearly 600 carotenoids in nature. In humans, four carotenoids (β-carotene, α-carotene, γ-carotene, and β-cryptoxanthin) have vitamin A activity, and they can be converted to retinal. Mature soybean seeds contain about 1 μg of carotenoids. However, concentrations of carotenoids are increased several fold during germination. Due to versatility of soybeans in use, genetic modification of soybeans is a popular subject. Genetically engineered soybean can now produce β-carotene 1,400-fold over non-trasngenic soybeans.
Vitamin E is a lipid-soluble compound and a family of eight related compounds that includes both tocopherols and tocotrienols. α-Tocopherol is most abundant in foods and also dominates in vitamin E supplements; the other leading types are β-, γ-, and δ-tocopherol. α-Tocopherol has become synonymous with vitamin E, and vitamin E is one of the most popular supplements. Vitamin E has antioxidant activities that stop the production of reactive oxygen species formed when unsaturated fatty acids undergo oxidations. Soybeans and corns contain γ-tocopherol (about 12.0 mg of γ-tocopherol in 100g of total soy lipids) (Fig. 5), while α-tocopherol is found in olive oil. In vitro experiments, γ-tocopherol killed animal cells at high concentrations, but α-tocopherol did not show cytotoxicities at the same concentrations. Interestingly, although people in the United State tend to use corn and soybean oil for cooking, most abundant in the body is α-tocopherol. Other than antioxidant activities, α-tocopherol is reported to inhibit protein kinase C activity, which is involved in cell proliferation and differentiation. Vitamin E inhibits platelet aggregation and enhances vasodilation. Vitamin E enrichment of endothelial cells downregulates the expression of cell adhesion molecules, thus decreasing the adhesion of blood cell components to the endothelium.
Synthesis of ergocalciferol from ergosterol (vitamin D2)
There are only a few food sources (fish, liver, and egg yolk) of vitamin D. These are many fortified foods (i.e. milk, soy drinks, orange juice and margarine) that contain vitamin D. Ergosterol, called provitamin D2, is found in ergot, yeast, and other fungi. It is converted to vitamin D2 (ergocalciferol) upon irradiation by ultraviolet (UV) light or electronic bombardment (Scheme 2), whereas, vitamin D3 (cholecalciferol) is normally synthesized in the human skin from 7-dehydrocholesterol. Vitamins D2 and D3 are about equal in activity in all mammals (some literatures described that vitamin D3 is slightly less bioactive). Deficiency of vitamin D can result in rickets (a softening of bones) in children and osteomalacia in adults. The relationship between ergosterol content in soybeans or soybean oils and soybean fungi was studied. For an example, in the studies of soybeans inoculated with spores of Aspergillus ruber, ergosterol concentrations in seeds increased with time of storage (Dhingra, et al. 1998).
Representative structures of soy phytoalexins
Phytoalexins are antimicrobial substances synthesized by plants that accumulate rapidly at areas of pathogen infection. They are, in general, broad spectrum inhibitors and structurally diverse molecules have been isolated from different plant species. Phytoalexins are known to inhibit bacterial or fungus cell wall biosynthesis, or delay maturation, or disrupt metabolism. To date, several soy phytoalexins have been reported. 6a-Hydroxyphaseollin was the first structurally defined phytoalexin isolated from fungal infected soybeans. Several other hydroxypterocarpan (benzopyrano-furanobenzenes) derivatives are biosynthesized as phytoalexins by soybean tissues on treatment with a variety of biotic or abiotic agents. Glyceollins are a family of pterocarpan found in the Fabaceae family including activated soy. They are biosynthesized from the isoflavone, daidzein (Fig. 1)\n\t\t\t\t\tvia 2\'-hydroxyisoflavone reductase as illustrated in Scheme 3. The concentration of phytoalexins in soybeans is very low since the compounds are only produced by soy as a defence mechanism from disease or infection. For an example, the accumulation of glyceollins in soybean cotyledon tissue was observed using four species of Aspergillus; 955 μg/g of glyceollins could be isolated from soybean cotyledon tissue inoculated with A. sojae. Representative phytoalexins identified from fungal infected soybeans are summarized in Fig. 8. Besides their antifungal or antibacterial activities, glyceollins have recently been demonstrated to be novel antiestrogens that bind to the estrogen receptor (ER) and inhibit estrogen-induced tumor progression (Zimmermann et al. 2010). Therefore, glyceollins may represent an important component of a phytoalexin-enriched food diet in terms of chemoprevention as well as a novel therapeutic agent for hormone-dependent tumors.
Biosynthesis of glyceollins
Coumestrol is classically categorized as phytoestrogens because this molecule binds to the estrogen receptor (ER). Coumesterol is originally isolated from alfalfa (Bikoff et al. 1957), but later soybeans and clover contain the highest concentrations of this molecule. Biosynthesis of coumestrol in soy is proposed based on the feeding experiments of labeled precursors to a coumestrol producing bacteria (Berlin et al. 1972). As illustrated in Scheme 4, the isoflavone, daidzein is reduced to form 2’-hydroxy-2,3-dihydrodaidzein which undergoes intramolecular condensation to yield 3,9-dihydroxypterocarp-6a-en. Biological oxidation of the dihydroxypterocarpen furnishes coumestrol.
Biosynthesis of coumestrol
Coumestrol has less estrogen activities than estrogen and therefore may reduce the risk of developing breast or prostate cancer in humans by preventing estradiol binding to estrogen receptor (ER). Coumestrol was reported to inhibit the enzymes involved in the biosynthesis of steroid hormone (aromatase and hydroxysteroid dehydrogenase), and inhibition of these enzymes results in the modulation of hormone production.
Representative structures of soyasaponins
Saponins are amphipathic glycosides grouped phenomenologically by the formation of soap-like froth when shaken in aqueous solutions. Structurally, saponins contain one or more glycoside moieties combined with a lipophilic triterpene derivative. Many health benefits of soybeans are believed to be attributed to their Saponins. Many soy products contain high levels of saponins. Raw soybeans contain between 2 and 5 g saponins per 100 g. Soy saponins are divided in two groups; group A saponins have and undesirable astringent taste, and group B saponins have the health promoting properties.
The blood cholesterol-lowering properties of dietary saponins are of particular interest in human nutrition. Saponins bind cholesterol and bile acids in the gut. Cancer cells have more cholesterol-type compounds in their membranes than normal cells. Soy saponins can bind cholesterol in vitro, and thus interfere with cell growth and division. Soy saponins also showed antifungal activities probably due to interference with cholesterol in fungus. Saponins cannot permeate the intestinal wall, but showed effectiveness in binding to cholesterol and making it unavailable for re-absorption within the small and large intestine. Apart from their important role in binding ability to cholesterol, soy saponins showed adjuvant effects for vaccines. An in vitro study demonstrated that soy saponins exhibited potent antiviral effects on the HIV virus. To date, over 26 soyasaponins have been isolated from soybeans and their gross structures were determined via high-magnetic field NMR or X-ray crystallography. Representative soyasaponins are illustrated in Fig. 9.
Lectins are plant derived proteins which are capable of binding to carbohydrate moieties of complex glycoconjugates but do not possess immunoglobulin nature. They typically agglutinate certain animal cells and/or precipitate glycoconjugates. Many members of the lectinic protein family agglutinate red blood cells. This particular nature of lectinic proteins is classified into hemagglutinin. Lectins are stable proteins that do not degrade easily. For examples, some lectins are resistant to stomach acid and digestive enzymes. Unfermented soy products contain high levels of lectins/hemagglutinins. Hemagglutinin renders red blood cells unable to absorb oxygen. However, the soybean fermentation process deactivates soya hemagglutinins, and thus the amounts of lectins present in soybeans have not been considered to be as potentially toxic components. On the other hand, some dried bean products may still contain a large amount of active lectins. These lectins are believed to trigger allergic reactions or toxic reactions in a person’s body. Person’s lectin sensitivity is largely due to 1) genetics, 2) a failure of mucosal immunity (secretory IgA), and 3) bacterial or viral infections that damage human cells, making human body susceeptable to lectin antibody/antigen reactions.
Water soluble vitamins isolated from soy
Soy contains several naturally occurring compounds that are toxic to humans and animals. The best known of the soy toxins is the trypsin (a serine protease found in the digestive system) inhibitors. In vivo studies using rat, high levels of exposure to trypsin inhibitors isolated from raw soy flour cause pancreatic cancer whereas moderate levels cause the rat pancreas to be more susceptible to cancer-causing agents. However, the US FDA concluded that low levels of soy protease inhibitors pose no threat to human health. Recently, a metalloprotein possessing toxicity to mice (LD50 7-8 mg/kg mouse upon intraperitoneal injection) was identified. This protein has a size of 21 kDa and was named soyatoxin. Some other biological properties of soyatoxin include hemagglutination and trypsin inhibitory activity.
Soybeans are not considered to be very rich sources of any particular vitamin, but they contain a wide variety of vitamins and do contribute to an overall nutritional well-being. Lipid-soluble vitamins (vitamin K, E, D and carotene) in soybeans are discussed above. The water-soluble vitamins in soybeans are thiamine, riboflavin, niacin, pantothenic acid, biotin, folic acid, inositol, choline, and vitamin C. Vitamin B6 was also reported to contain in soybeans. Thus, soy includes essential vitamins except for vitamin B12 and E. However, soy contains a vitamin E precursor, carotene. Numerous studies have been conducted over the past decades on the relative distribution and concentrations of these vitamins in different portion of soy. The cotyledons contain notably greater amount of all water soluble soy vitamins than those in the hypocotyl. The quantity of all vitamins except thiamine increases through germination (Wai et al., 1947).
This chapter summarizes structures of biological active molecules isolated from soy, and their biological activities are briefly reviewed. Since the discovery of soybeans as rich source of protein and oil in 1904, a numerous number of experimental data have been accumulated on chemistry and biochemistry of phytochemicals isolated from soy and soy products. To date, a wide variety of organic compounds have been characterized from soy. The interest of structure elucidation studies of bioactive molecules in soy was to obtain insight into correlation between the reported health benefits, which are associated with soy intake, and soy phytochemicals. Thus, many structural studies on soy have not aimed to discover novel molecules, albeit these efforts resulted in discovery of complex soyasaponins (Fig. 9.). The consumption of soy products has many health benefits, including protection against breast cancer, prostate cancer, menopausal symptoms, heart disease and osteoporosis. These health benefits of soy are believed to be due in part to phytoestrogenic activity of isoflavones, which are stored as glucosyl- and malonyl-glucose conjugates in soybeans. Isoflavon-glucosyl conjugates show very poor oral bioavailability. Recently, the negative aspects of soy have been reported (3.1.8). However, the controversy reports about the adverse effects of soy are not clear. This may be due to the lack of understanding of metabolism and bioavailability of isoflavone-glucosyl conjugates in soy. Some researchers concluded that there is little clinical evidence to suggest that isoflavones will cause the adverse effects (e.g. breast cancer risk in healthy women). It is important to note that all phytochemicals isolated from soy show an array of weak biological activities, and thus, normal consumption of foods that contain these phytochemicals should not provide sufficient amounts to elicit a visible physiological response in humans in short-time clinical researches.
Unfermented soy products contain high levels of lectins/hemagglutinins, and very low level of soy toxins (protease inhibitors). Most of these proteins will be deactivated through food processing. Remarkably, seeds of soy contain very high levels of protein, carbohydrate conjugates, oil, and minerals. On the other hand, remaining components discussed in this chapter can be isolated minute quantities from soybeans, however, a wide range of health benefit of soy phytochemicals (lipids, phytoestrogens, soy sterols, vitamins, soyasaponins) contributes to the overall nutritional well-being of humans.
I would like to thank Emeritus Professor Isao Kitagawa for valuable discussions for this document.
A vast majority of microorganisms in the world exist within biofilms, which are weak hydrogels that often form at various interfaces [1]. The biofilms consist of up to 98% water, and they are typically composed of polymicrobial aggregates that are encased in extrapolymeric substances (EPS) [2, 3, 4, 5]. Besides acting as a protective barrier, the EPS, which is made of DNA, proteins, and polysaccharides, aid in adhesion and water retention [4]. Pseudomonas aeruginosa is a well-known opportunistic human pathogen that is a common cause of hospital-acquired infections in burn wounds and eyes [6, 7, 8], and it is known to create persistent infection in cystic fibrosis (CF) patients [8, 9, 10, 11, 12, 13, 14, 15], having resistance to many classes of antibiotics [16, 17, 18]. PAO1, a medically-relevant strain of P. aeruginosa that is used in this study, acts as the model for biofilm-forming bacteria. To grow, bacterial cultures need water, a source of carbon, a source of nitrogen, and trace amounts of salts. The lysogeny broth (LB) is a complex and non-selective medium; many different types of bacteria can grow on non-selective medium. Lysogeny broth was formulated by Giusseppe Bertani in 1951 to optimize Shigella growth, but it has since become the standard for growing many bacterial cultures [19]. Lysogeny broth is composed of: 1% tryptone (source of amino acids); 0.5% yeast extract (source of vitamins, amino acids, nitrogen, and carbon); [20] and 1% NaCl (provides osmotic balance) [21]. Yeast extract is made from baker’s yeast (Saccharomyces cerevisiae) grown to a high concentration and then exposed to high temperature or osmotic shock, killing the yeast and starting autolysis of the cells through the yeast’s own enzymes [20, 22, 23]. The resulting extract solution is further filtered and spray-dried into a powder [20]. Proteins make up the most significant component of the powdered yeast extract at 62.5–73.8 wt% [20]. The average molecular weight of the yeast extract is 438 Da with 59.1% of the total under 300 Da [23]. Using additions of glycerol, glucose, sucrose, sodium chloride (NaCl), and silver nitrate (AgNO3), this paper investigates various modifications of the LB medium for their effects on the biofilm.
Both the biofilm’s structure and the cell-to-cell communication mechanism of the bacteria, known as quorum sensing (QS), are affected by their environment and the medium composition [24]. Quorum sensing controls additional properties that influence biofilm structures of bacteria, such as the production of extracellular DNA, proteins, mucus, and lipids [24, 25, 26]. When the growth environment becomes more viscous through the addition of glycerol, strains of Pseudomonas produced high-molecular-weight EPS and developed more robust biofilms [27]. The nutritional condition, such as the carbon source, influences the QS-associated swarming motility of P. aeruginosa [25]. While glucose supplementation limits bacterial motility, producing scattered, mushroom-like microcolonies, increasing the concentration of glucose from 0 to 2.7% caused an increase in the overall formation of biofilm [24, 25, 28, 29].
High osmolarity had a detrimental effect on biofilm of P. fluorescens, at roughly 0.4 Osm L−1 of either NaCl or sucrose, and the formation of biofilm decreased by four-fold as compared to lower concentrations of each component [30]. Similarly, mutant strains of P. aeruginosa that are found in CF patients transition from a non-mucoid to an alginate-overproducing state under osmotic stress that is induced by concentrations of 0.2–0.5 M NaCl (~1.2–3%) or 10% sucrose [31]. Silver has broad-spectrum antimicrobial effects on gram-negative bacteria that are well-documented [32, 33]. For instance, for concentrations of silver sulfadiazine that are lower than 0.16 μg mL−1, planktonic growth of P. aeruginosa was unchanged; however, at or above this threshold amount, the concentration of the planktonic bacteria was reduced by five orders of magnitude [34]. Silver sulfadiazine was even effective against mature biofilms above a threshold dose of 1 μg mL−1, and at concentrations of 10 μg mL−1, it can completely eradicate a pre-established biofilm of P. aeruginosa [34].
The following sections of this study cover three different methods of characterizing biofilms: (i) rheology to quantify the impact of the modified medium on the mechanical strength of the biofilm; (ii) ferning to characterize the mass transport of the salts through the polymer matrix of the biofilm during desiccation; and (iii) birefringence to observe self-assembly behavior of the solute in the biofilm.
The study of flow and deformation of matter (rheology) enables characterization of its structure and mechanical properties. Rheology is an especially valuable tool for understanding a vast range of “soft matter” that falls between liquid and solid phases [35]. Soft matter can be divided into four classes: (1) polymers, a long repeating chain of monomers which for biological samples include proteins, DNA, and cellulose; (2) colloids, a large category of materials that describe a suspension of one material into another medium such as aerosols, foams, emulsions, suspensions, and pastes; (3) amphiphiles, molecules with dual characteristics where one end of the molecule likes the solvent (hydrophilic), while the other end does not (hydrophobic) include surfactants that are amphiphiles at the air-water interface; and (4) liquid crystals, rod or disk shaped molecules that self-assemble to form orientation order but not positional order, resulting in an anisotropic fluid [35, 36].
Rheological techniques can characterize the strength and behavior of clinically relevant biological fluids such as mucus, blood plasma, and bacterial biofilm. More importantly, we can also use rheological measurements to drive the treatment of the biofluids toward a favorable clinical outcome. A rheological testing can quantify the viscous and elastic properties of a material. Two main modes of testing exist on a rotational rheometer: (1) steady-shear testing mode (Figure 1a–d), where the material is sheared between a stationary bottom plate and rotating top plate at a given stress or strain; and (2) the oscillation mode (Figure 1e–g), where the top plate oscillates back and forth at a set frequency and amplitude.
Shear and oscillatory rheological techniques. (a) In a two-plate steady-shear system where the top plate is moving, the velocity (v) of the fluid is dependent on the gap height (h). (b) Samples can exhibit several different flow behaviors of stress versus strain rate, including (1) ideally viscous Newtonian fluid; (2) shear-thinning fluid; (3) shear-thickening fluid; and (4) yield stress fluid. Yield stress materials have a minimum stress (τy) that must be overcome before flow starts. (c) The stress-strain curve demonstrates a material with (2) no yield stress and (4) a material with a clear yield stress calculated using the tangent crossover point method. (d) The viscosity-shear rate plot shows (1) Newtonian fluid; (2) shear-thinning fluid with no yield stress reaching zero-shear viscosity (η0); (3) shear-thickening fluid; and (4) shear-thinning fluid with yield stress. (e) In a two-plate system the top plate can oscillate back and forth at set amplitude or frequency for oscillatory rheology tests. The amplitude sweep test has constant frequency (ω0) with changing strain amplitude while the frequency sweep test has constant strain amplitude (γ0) with changing frequency. (f) During the amplitude sweep test, the strain values up to the limit of the strain where the G′ and G″ values are constant (γL) are called the linear viscoelastic region (LVR). The point where G″ crosses over G′ is called the flow point (γf) [72]. (g) In a frequency sweep, when G′ > G″ materials are said to be solid-like and when G″ > G′, materials are said to be liquid-like.
From the shear-flow sweep test, the stress (τ) versus shear rate (
Two main types of oscillatory testing exist (Figure 1e–g). An amplitude sweep test oscillates the upper plate back and forth at a set frequency (ω0) at increasing strains (Figure 1e). On the modulus (G′—elastic; G″—viscous) versus strain plot (Figure 1f), the plateau is the linear viscoelastic region (LVR), and the strain limit of the region is γL. A frequency sweep test oscillates at a set amplitude (γ0), which has been determined previously from the amplitude sweep test to be within the LVR, at increasing frequency (Figure 1e). The frequency sweep describes how the material acts when the material is stressed for different periods. For example, when Silly Putty is stressed quickly by throwing it on the floor, it bounces back, acting like a rigid solid. However, when the Silly Putty sits at rest and experiences low stress over a long period of time, it spreads out, acting like a viscous fluid. The frequency sweep (Figure 1g) reveals if the material is solid-like (G′ > G″) or liquid-like (G″ > G′) and if the behavior is frequency dependent (G′(ω), G″(ω)) or independent. Stable gels and suspensions are typically solid-like and frequency-independent, so these types of materials are called “gel-like.”
Previous studies on biofilm rheology using various techniques of rheological measurement have found the elastic modulus (G′) to range in order of magnitude from 10−2 to 104 Pa for bulk biofilms at solid-liquid interfaces using plate-on-plate methods, while the values of yield stress (τy) range in order of magnitude from 10−1 to 105 Pa [2, 37, 38, 39, 40, 41, 42]. The wide-ranging values of G′ and τy in the literature reflect the variability in the compositions of the biofilms, diversity of growth mediums, variability of growth conditions, and most importantly, natural variability of response of the microorganisms, even to the same medium and growth conditions. This chapter uses the techniques established in our previously published work on the non-destructive development and characterization of rheological properties of biofilms [43]. Using this non-destructive method, the measured values of elastic modulus and yield stress of PAO1 that were grown in standard LB medium were both between 0.1 and 10 Pa [43].
Biological fluids like tears, cervical mucus, and saliva are all shown to self-assemble into fractal-like patterns of crystallization when they are dried [44, 45]. A fractal is a structure that is made of smaller parts that resemble the bigger parts, with a high degree of organization and self-similarity. This structure can be characterized with a specific fractal dimension [44]. Fractal dimension is a measure of complexity of the fractal pattern [46]. Random nucleations of salts initiate the process of crystallization, where its growth is limited by the diffusion of salt through the polymer matrix (proteins or macromolecules) [47]. Therefore, the combined effects of ionic strength, osmolarity, and the size and concentration of macromolecules control the behavior of crystallization, where too little or too much of one factor can dramatically alter the pattern of crystallization [48, 49]. A typical crystallization of biosaline proceeds in the following manner: (i) salt nucleation initiates the process of crystallization; (ii) the nucleation point grows with some symmetry into a highly-branched structure whose growth is modified by the interaction of the salt with the biological matter; (iii) the branches do not overlap or merge [47, 50, 51]. The process of crystallization of biofluids is called “arborization,” “ferning,” or “dendritic growth” in various literature [45]. In this paper, the general formation of salt crystals will continue to be called crystallization, while the specific crystallization of the biofluids that result in fractal patterns will be called ferns.
The ferning patterns of the dried samples of tears and saliva have been used for years as a supplementary diagnostic tool [49, 52]. The ferning patterns in saliva and in tears exhibit different morphologies; saliva produces linear ferns with branching angles of 90°, while ferns from tears have more curvature with tightly packed branches with acute angles. Ferning patterns from tears and saliva are traditionally classified in a qualitative manner according to Rolando’s system as Type I to Type IV [53]. Type I has the most ferning and the highest concentration of protein, while Type IV has no ferning and the lowest amount of protein [49]. Samples of tears from healthy individuals typically exhibit robust, highly-branched ferning patterns (Type I and II), while samples from patients with eye or immune diseases show little to no ferning (Type III and IV) [48, 49, 52]. Through analysis of X-ray microscopy and scanning electron microscopy (SEM), the molecular structure of the ferns from tears is revealed to be composed of NaCl, KCl, and proteins [48]. In addition to helping detect infection, saliva ferning pattern has been shown to be useful for tracking ovulation cycle from highest fertility level during estrus to lowest fertility level during diestrus [54].
Cervical mucus is a heterogeneous hydrogel that changes over the course of an animal’s reproductive cycle [44, 45, 55]. Regardless of the source, human or otherwise, high levels of estrogen are produced during ovulation or peak fertility, resulting in linear ferns with branching angles of 90°, while no ferning is found during the period of low fertility when progesterone is dominant [44, 45]. During ovulation, the cervical mucus is over 98% water with its highest level of salt while both water content and salt content drops during low fertility period [45]. The low salt content during low fertility period is the cause of the lack of ferning pattern.
SEM analysis of ferns from gelatin-NaCl mixture revealed that the backbone of the ferning pattern was a series of interlocking crystalline blocks that were 10–30 μm in size [47]. When the fractal dimension of bovine cervical mucus (BCM) that was taken during ovulation was determined using the box-counting method, it was about 1.7, characteristic of diffusion-limited growth processes [44, 46]. Box counting method is based on counting non-empty boxes making up a fractal pattern on a grid [46]. A diffusion limitation was observed with the gelatin-NaCl mix as well. The ferning pattern became much less geometric and increasingly random at higher gelatin-to-NaCl ratios, where more diffusion limitation occurs due to the crosslinking of the gelatin, and the ferning ceased at extremely high gelatin-to-NaCl ratios [47]. Furthermore, the ferning pattern developed curvatures at evaporation rates above a threshold value of 11 μm s−1 [47].
Bacterial biofilms produce ferning patterns that are similar to gelatin and mucus samples [50, 56]. Upon evaporation of droplets of solutions of various salts with cells of E. coli and Bacillus subtilis, ferning patterns emerged where the crystallized top layer covered a base layer that consisted of bacterial cells. The structure of the E. coli ferns was linear with branching angles of 90°, similar to cervical mucus. Neither sterile saline solutions nor E. coli in pure water produced ferning, confirming the previous findings that ferning results from balanced proportions of salts and macromolecules [50, 56]. Bacteria inside the crystalline structure were effectively in a state of suspended animation that was capable of reanimation after rehydration, even a week later [50]. This crystallization was hypothesized to be a form of biomineralization [50], which occurs when biological organisms produce organo-mineral hybrids that give the organism mechanical strength and hardness. Examples of biomineralization that are found in nature include bones, teeth, shells, corals, and algal silica [57]. Previous studies on strain PAO1 of P. aeruginosa in flow cell reactors have shown biomineralization of calcium carbonate within the EPS of the biofilm [58]. SEM of the ferning sample of E. coli revealed a 3D structure that was composed of dried EPS, bacteria, and salts, with the salts concentrated in the crystalline region, consistent with the previously mentioned studies [50].
Studying the ferning pattern and complexity of biofluids or biogels gives a simple and indirect measurement of the structures within the material that guide or hinder the movement of ions that ultimately form these distinct crystallization patterns. While much of the ferning patterns seen in biofluids are linear patterns with 90° branching angles, tightly packed and curved ferning patterns can be expected to develop in environments that induce fast evaporation such as in low-viscosity fluids or environments that are highly diffusion limited such as in high macromolecule to salt ratio fluids.
One of the techniques of self-assembly for small particles is through depletion attraction in a solvent during solvent evaporation [59, 60]. Depletion attraction is an entropic force that becomes relevant when the particles in the solvent move close enough together that their excluded volumes overlap [61]. This overlap increases the osmotic pressure in the surrounding fluid and further pushes the particles together [59]. These highly ordered or anisotropic solution is described as having a liquid crystal phase and this phase is birefringent, which means that their ordered state will split light into two beams with perpendicular polarization [36, 60, 62]. Liquid crystal phases have been observed with many different types of biopolymers such as DNA, peptides, glycopolymers, proteoglycans, viruses, collagen, cellulose, phages, and chitin [60, 61, 63]. Liquid crystals form: (i) nematic phase where the molecules form directional order but no positional order; (ii) smectic phase with positional order; or (iii) chiral phase with twisting order [60]. Of these, biopolymers most commonly have nematic phase.
P. aeruginosa that exists in a viscous or anaerobic environment is stimulated to transcribe filamentous Pf bacteriophages that are about 2 μm in length and 6 nm in diameter [64]. In P. aeruginosa biofilm, the filamentous phage self-assembles through depletion attraction, with the biopolymers exerting the osmotic force that bundles the phage strands. These highly ordered anisotropic regions of nematic phase liquid crystals are birefringent, possessing a large negative charge, and the anisotropy was shown to increase with the ionic strength and the molecular weight [64, 65]. Birefringence is not only a direct indicator of molecular order, but it is an indicator for P. aeruginosa biofilm strength, surface adhesivity, desiccation tolerance, and antibiotic resistance [62, 64]. The filamentous bacteriophages facilitate chronic infection of P. aeruginosa in the host by promoting a less invasive, less inflammatory but more resistant, more persistent form of P. aeruginosa [66]. In addition, Pf phages can bind iron to inhibit the metabolic activity of other pathogens such as Aspergillus fumigatus [67].
Liquid crystal methods provide the means to study the structure and behavior of filamentous bacteriophages without perturbation [65]. Moreover, liquid crystal analysis, specifically through detection of its birefringence, was used to detect analytes such as glucose, cholesterol, E. coli, and even viruses such as Ebola and HIV [68]. This detection method was made possible through enzymatic reaction in response to analytes within the mesophase of the normally optically isotropic lipidic cubic phases that results in the formation of strongly birefringent liquid crystal phases that are easily detected optically [68]. The exogenous and endogenous birefringence from various classes of analytes were exploited to make simple and cheap detection tool that was proposed as a new diagnostic tool that can be utilized in industry or in the field to detect biothreats [68].
Biofilm is composed of motile bacterial cells, non-motile bacterial aggregates, and mucoid hydrogels of EPS that have a heterogeneous, highly-porous microstructure, allowing diffusion of water, nutrients, waste, and electrolytes [26, 69]. A complex set of interactions between the electrolytes, solutes, bacteria, and biopolymers dictate the strength, bacterial resistance, and infection persistence of the biofilm. The objective of this study is to characterize the behavior of the bacterial culture in the presence of various environmental conditions, including a highly viscous media, nutrient-enhanced media, high osmolarity media, and antimicrobial media. The interaction of the bacterial culture with its nutrient environment is measured as a function of the strength of its biofilm through rheological analysis, while its ferning pattern characterizes the mass transport through the environment in the biofilm. Additionally, birefringence inside a biofilm provides insight into the solute interaction with the biofilm.
The strain of Pseudomonas aeruginosa that was used for the entire study was the laboratory-adapted wild-type strain, PAO1. Miller lysogeny broth (LB) was prepared from BD Difco dry powder and autoclaved. Five different types of chemical modifications were made to the lysogeny broth: (i) glycerol was added to form between 1 and 15 v/v% in LB medium; (ii) glucose was added to form concentrations between 0.5 and 4.5 w/v% in LB; (iii) sucrose was added to form concentrations between 0.5 and 4.5 w/v% in LB; (iv) NaCl added to form concentrations between 1.5 and 5 w/v% in LB; and (v) AgNO3 added to form concentrations between 0.001 and 1 mM in LB. Modified LB medium in a petri dish (3.6 mL) was inoculated with an overnight culture of PAO1 (0.4 mL) and was incubated for 6 days at 37°C. Some of the dishes of modified LB medium were kept sterile and incubated along with the biofilm samples to act as a negative control.
The sample rheology was measured on the Discovery Hybrid Rheometer 3 (DHR3, TA Instruments, USA) using a 40-mm stainless steel plate geometry at 25°C. The measurements took place in the following order:
Pre-stressed: 0.1 Pa, 2 minutes
Frequency sweep: γ0 = 0.1 (biofilm), γ0 = 0.005 (sterile LB), ω
Stress sweep: τ
Detailed description of the sample inoculation, the incubation, and the rheological measurement methods are located in our previous work [43].
The biofilm samples were dried in the incubator, forming ferning patterns that were large enough to be easily seen by the unaided eye. In this paper, the previous qualitative method for ferning characterization was converted to a quantitative method of image analysis by calculating the coverage area, the fractal dimension, and the complexity score (degree of branching) of the ferning pattern. This analysis was completed by taking photographs of the surface of the petri dish, converting the photographs to black and white image on MATLAB (Figure S4,
Microscopic images of the biofilm in its liquid and its dried ferning state were taken using an Eclipse Ti-S inverted microscope (Nikon, Japan). The polarized images were produced with polarized filters.
Data from the sweeps of frequency showed the difference in the viscoelasticity of the samples of biofilm (filled square) and the samples of sterile LB medium (unfilled circle) that were incubated for 6 days (Figure 2). For brevity, the term “unmodified LB” will refer to the standard LB medium without chemical alterations, while “modified LB” will refer to any of the five chemical additions to standard LB medium (glycerol, glucose, sucrose, NaCl, and AgNO3). The sweep of frequency of the biofilm showed frequency-independent, elastic modulus (G′) dominance over the viscous modulus (G″) for all of the samples, as expected for a weak gel (Figure S3,
Modulus |G*| calculated from the sweep of frequency showing the biofilm (filled square) and sterile LB medium (unfilled circle) samples. The results of the (a) sweep of frequency from ω ∈[0.1, 1] rad s−1 (biofilm γo = 0.1 and sterile LB medium γo = 0.005) of biofilm samples that were grown in unmodified LB medium and of sterile unmodified LB medium. The mean modulus |G*| was 0.015 Pa for biofilm and 0.0015 Pa for sterile unmodified LB medium. (b–f) The mean modulus |G*| is plotted (red) with relative errors that were calculated from standard error to ensure symmetry (n ≥ 3). The average |G*| of biofilm that was grown in modified LB medium with the following concentrations (b) 1–15 v/v% glycerol, (c) 0.5–4.5 w/v% glucose, (d) 0.5–4.5 w/v% sucrose, (d) 1.5–5 w/v% NaCl and (e) 0.001–1 mM AgNO3 are shown. The gray regions in (b) and (f) represent inhibiting concentrations that had no biofilm growth.
The values of yield stress were derived from the experiments of increasing strain, where the yield stress is the point of offset of a stress-versus-strain curve. The stress-versus-strain data of the unmodified LB biofilm (filled square) and sterile unmodified LB samples (unfilled circle) showed that the samples of biofilm exhibited an appreciable yield stress (Figure 3a). After yielding, the material stress was constant (<1 Pa) at high strain, while the samples of sterile medium demonstrated no yield stress. In fact, none of the sterile modified or unmodified LB mediums had an appreciable yield stress (Figure S2,
(a) The stress versus strain data of biofilm that was grown in unmodified LB medium (filled square) and of samples of sterile unmodified LB medium (unfilled circle). (b–f) For the mean yield stress of biofilm that was grown in unmodified LB medium (red square), the standard error bars were converted to relative errors to ensure symmetry on the y-axis (n ≥ 7). Plots of yield stress τy of the biofilms that were grown in modified LB medium at the following concentrations (b) 1–15 v/v% glycerol, (c) 0.5–4.5 w/v% glucose, (d) 0.5–4.5 w/v% sucrose, (e) 1.5–5 w/v% NaCl and (f) 0.001–1 mM AgNO3 are shown. The mean τy was 0.32 Pa for the unmodified LB biofilms, while the LB medium did not have a yield stress. The gray regions in (b) and (f) represent inhibiting concentrations that had no biofilm growth.
With the addition of glycerol (0–15 v/v%), the complex modulus of the biofilm increased by almost an order of magnitude between 0 and 2% glycerol, remained constant between 2 and 10%, and experienced a dramatic drop in modulus for concentrations greater than 10% to modulus values that are comparable to sterile LB (Figure 2b). The yield stress of the biofilm showed similar trends, increasing by one order of magnitude with glycerol from 0 to 10% until concentrations of glycerol that were greater than 10% impeded the growth of biofilm, also resulting in no yield stress (Figure 3b). The addition of glycerol increased the viscosity of the medium as well as inducing high osmolarity (1.4 Osm L−1 at 10%), promoting stronger biofilm. Other studies with glycerol-supplemented medium saw an increase in the production of EPS by biofilm, consistent with the present study [27, 70]. The glycerol can trigger pathways of production of EPS; [27] however, at high concentrations of glycerol, the diffusion-limiting environment of the highly viscous solution with high osmotic pressure (>4 Osm L−1 at >10%) appeared to inhibit growth. The complex modulus of the modified LB medium, on the other hand, stayed relatively constant with glycerol addition. The dramatic drop in the modulus of biofilm samples that were grown in medium that was modified with >10% glycerol corresponded with an apparent lack of biofilm in the Petri dishes, as the dishes appeared clear and yellow instead of opaque and greenish (Figure S1,
The modulus of the biofilm increased by one order of magnitude by increasing the concentration of glucose from 0 to 4.5% (Figure 2c), indicating that glucose was being utilized by the bacteria as an additional source of carbon which promoted growth and development of a stronger network of biofilm. The rheological results of the sterile glucose-modified LB medium did not change significantly from the unmodified LB medium. The values of yield stress followed the same trend, where the biofilm that was grown in glucose-modified LB medium had yield stresses that were an order of magnitude larger than the unmodified LB biofilm (Figure 3c). A previous study observed the same effect, finding that the addition of glucose up to the highest level tested, which was 2.7%, enhanced biofilm production [29]. The maximum addition of glucose (4.5%) induced osmotic pressure of 0.25 Osm L−1, which did not cause inhibiting effects.
Based on the rheology, sucrose did not increase biofilm production, as no change existed in the modulus (Figure 2d) or yield stress (Figure 3d) of the biofilm. In previous studies, concentrations of sucrose above 10% in medium for P. aeruginosa resulted in biofilm with mucoid development, while P. fluorescens started to experience adverse effects above 15% at which point the biofilm dramatically decreased [30, 31]. In those studies, bacterial culture reached an inhibiting level of sucrose at 15% due to osmotic pressure (0.44 Osm L−1) [30]. In the present work, samples of PAO1 experienced a maximum of 0.13 Osm L−1 in osmotic pressure from modification with sucrose, which is well below the reported osmotic level for inhibition. P. aeruginosa may not be capable of utilizing sucrose, so in contrast to the simpler glucose, sucrose had little impact on the rheological properties of the biofilm.
Unmodified LB medium already consists of sodium chloride (NaCl) at a concentration of 1%, and the modified concentration varied from 1 to 5% of NaCl. The complex modulus of biofilm remained constant for concentrations below 2.5% and increased by one order of magnitude for concentrations between 2.5 and 5%, while the modulus of the sterile modified LB medium was not affected by the concentration of NaCl (Figure 2e). Similarly, the yield stress increased as the concentration of NaCl was increased greater than 2.5% (Figure 3e). NaCl is already required for bacterial growth to provide osmotic balance, but a larger amount of salts appeared to promote stronger biofilm. The change in the biofilm could be caused by the higher salinity or osmolarity, making the environment hostile, triggering a higher level of production of alginate and other types of EPS as a countermeasure. Previous studies found that concentrations of NaCl between about 1 and 3% increased production of biofilms in S. aureus and P. aeruginosa, while concentrations of about 6% of NaCl prevented growth of biofilm in S. aureus [29, 31]. At concentrations of NaCl above 10%, no biofilm growth was observed, and the plate quickly crystalized to cubes of salt (Figure S6,
Silver has antimicrobial properties that can inhibit bacterial growth and development of biofilm. Supplementation of silver nitrate (AgNO3) to the modified LB medium has no impact on the complex modulus (Figure 2f) or the yield stress of the biofilm for concentrations below 0.1 mM (Figure 3f). Past this concentration, the modulus instantly reduced to the same level as the sterile modified LB medium, and the yield stress disappeared. Correspondingly, the plates of biofilm at the higher silver concentrations appeared clear and less viscous, resembling sterile modified LB medium (Figure S1,
The rheological parameters of elastic modulus and yield stress are useful measures of the strength of a biofilm. The complex modulus and the yield stress of the biofilms increased with the addition of glucose, which served as an additional source of carbon, but they were unaffected by addition of sucrose, which is a complex sugar that the bacteria could not utilize. The strength increased to an extent with osmolarity (glycerol and NaCl) and dramatically reduced to their sterile baseline at concentrations that were higher than the inhibitory threshold of an antimicrobial agent (AgNO3). Samples with higher rheological properties correlated with a biofilm that appeared more viscous than the unmodified LB biofilm, while samples with lower modulus, and lacking a yield stress, such as high concentrations of glycerol and silver ions, appeared less viscous and free of biofilm. The values of modulus and yield stress for the samples of biofilm displayed the same medium dependent response; therefore, either measurement would be a useful metric of the strength of biofilm. Out of the five chemical modifications, three modifications increased the strength of the biofilm when compared to the unmodified LB biofilm: glycerol for concentrations up to 10%, glucose for concentrations at least up to 4.5%, and NaCl for concentrations higher than 2.5%. One of the chemicals, sucrose, had no measurable effect on the strength of the biofilm for concentrations at least up to 4.5%, while another modifier, AgNO3, inhibited bacterial growth at a concentration above 0.1 mM.
After the plates were dried over a span of weeks in the incubator, they were photographed, and the photographs were converted to black and white images and cropped (Table S1,
A guide for the ferning complexity score of the dried biofilm ferning pattern.
The ferning coverage was calculated quantitatively based on the percent of white pixels in the black and white images that were converted from its original photograph (Figure 5a). A photograph of the ferning on a plate of unmodified LB biofilm showed high ferning coverage (top photos), while the plate of sterile unmodified LB medium was noticeably absent of ferning with low calculated coverage (bottom photos). Even without biofilm growth, the sterile coverage values were not zero because the lighting and the glare of the plate surface produced some pixel artifacts. Figure 5b–f shows the mean coverage of the plates of unmodified LB biofilm (red filled square, n = 12) and of the plates of sterile unmodified LB medium (red unfilled circle, n = 7) plotted with the data of the modified LB medium. The left black y-axis is the ferning coverage, while the right gray y-axis is the qualitative ferning complexity score for biofilm (gray filled square) and the sterile LB medium (gray unfilled circle).
(a) The original photograph was converted to a black and white image before the ferning coverage was calculated from the processed image. An example of ferns from biofilm that was grown in unmodified LB medium with 46.2% ferning coverage and from a plate of sterile unmodified LB medium showing zero ferning coverage. (b–f) Ferning coverage (left y-axis in black) and complexity score (right y-axis in gray) of biofilm that was grown in modified LB medium (coverage: black filled squares; complexity: Gray filled squares) and of sterile plates (coverage: black unfilled circles; complexity: Gray unfilled circles). The mean coverage and standard deviation of biofilm that was grown in unmodified LB medium (n = 12) and in sterile unmodified LB medium (n = 7) are plotted in red across figures (b–f). The results of the biofilm that was grown in modified LB medium with (b) glycerol, (c) glucose, (d) sucrose, (e) NaCl and (f) AgNO3 are shown. The change in morphology of the biofilm ferning pattern with (b) glycerol at concentrations of 2% (top left) and 10% (top right); (c) glucose at concentrations of 0.5% (top left) and 4.5% (top right); (d) sucrose at concentrations of 0.5% (top left) and 4.5% (top right); (e) NaCl at concentrations of 1.5% (top left) and 5% (top right); and (f) AgNO3 at concentrations of 0.001 mM (top left) and 1 mM (top right) are shown. The gray regions in (b) and (f) represent inhibiting concentrations that had no biofilm growth.
With the addition of glycerol, the ferning pattern of the samples of biofilm initially changed from a complexity score of 5 and a coverage of 47% (unmodified LB biofilm) to a complexity score of 8 (Figure 5b). The change in ferning morphology from the orthogonal form to the acute branching form occurred at the lowest tested concentration of glycerol (Figure 5b: 2% plate, top left). However, as the concentration of glycerol increased, the ferning coverage dropped dramatically, reaching zero at around 8%. From both the visual inspection and the rheological measurement, samples below 10% had strong biofilm. However, a large amount of glycerol prevented the sample from completely drying, leaving the surface of the sample looking shiny and wet, causing both the ferning coverage and the complexity score to drop between 4 and 10% glycerol (Figure 5b: 10% plate, top right). The plates with concentrations of glycerol above 10% never dried, so no photographs were taken, and values of the coverage and complexity score were assumed to be zero. Sterile LB medium coverage and complexity score did not change from the unmodified values of about zero.
In samples that were modified with the addition of glucose, the ferning coverage remained consistent, while the complexity score changed from 5 to 8 at 2.5% and then held steady at higher concentrations of glucose (Figure 5c). The pattern on the plates transitioned from standard orthogonal ferning at low concentrations of glucose (Figure 5c: 0.5%, top left) to acute branching at high concentrations of glucose (Figure 5c: 4.5%, top right). The coverage and the complexity score on the sterile plate remained unchanged from the standard values.
The desiccated plates of medium modified with sucrose had the most unusual patterns (Figure 5d). Both the coverage (47–30%) and the complexity score (5–3) dropped when the concentration of sucrose increased from 0 to 2%. However, with further increase from 2 to 4% sucrose, the coverage increased to 50%, while the complexity score continued to show less degrees of branching. Similar to glycerol, sucrose is hygroscopic, so plates appeared shinier and somewhat wet with increasing concentration of sucrose. At the same time, the ferning on the surface evolved (Table S1,
With further addition of sodium chloride (NaCl) to the modified LB medium, the ferning coverage remained consistently around 50%, and the complexity score remained around 5 (Figure 5e). Still, a change to the pattern exists, as the ferns evolved from thin branches (Figure 5e: 1.5%, top left) to a more pronounced branching with large crystalline formations with increasing concentration of NaCl (Figure 5e: 5%, top right). While the ferning branches became wider with the further addition of NaCl, the complexity score did not change. The orthogonal morphology and the non-overlapping crystallization appear to naturally limit the maximum coverage of the ferning pattern, resulting in a consistent 40–50% coverage. The sterile dishes with modified LB medium that contained the same amount of salt did not produce ferning patterns, so the coverage and complexity score remained zero.
The complexity score for the plates that were treated with silver nitrate (AgNO3) remained 5 for concentrations below 0.1 mM, but at higher concentrations, no biofilm growth occurred, resulting in a complexity score of 1 (Figure 5f). Even with no measurable biofilm, the plates contained clusters of dried materials that resembled nucleation points (Figure 5f: 1 mM, top right). For concentration below 0.1 mM of silver nitrate, the ferning patterns were orthogonal (Figure 5f: 0.001 mM, top left), and the coverage was in the range of 30–40%. Finally, the coverage dropped below 10% at concentrations that were greater than the inhibiting concentration of 0.1 mM. The coverage did not completely drop to 0, as beige clusters were left behind on the plate, which was the same reason that the complexity score was 1 for the highest concentration even though no biofilm was present.
Similar to the dependence of rheological properties of the biofilm on the nutrient environment, the ferning pattern was dependent on the properties of the bacterial biofilm, so it changed with the composition of the medium. No ferning existed on plates that lacked biofilm. The presence of biofilm, confirmed rheologically and visually, correlated with robust ferning patterns. Using the same box-counting method, ferning patterns on the samples of unmodified LB biofilm had a fractal dimension of 1.8 (Figure S5,
The complexity score and the ferning coverage was higher for stronger biofilms (higher G* and τy) that caused more limitations in mass transfer, and both values dropped to nearly zero when no biofilm was present. The morphology of ferns with a complexity score of 8 that were produced by the biofilms with higher elasticity was similar to random/acute-angled branching ferns that were produced under conditions of increased diffusion limitation in a previous study [47]. Exceptions were the plates that appeared to never fully dry due to the high concentration of hygroscopic materials like sucrose or glycerol. So, even as the rheological properties of the biofilm increased (glycerol) or stayed constant (sucrose), the complexity score and coverage dropped with increasing amounts of the modifying chemical. The ferning coverage never exceeded 60%, indicating a natural growth limitation that was based on the available space and the morphology of the ferns. The videos of the ferning process demonstrated how these branches quickly started and stopped growing without any of the branches overlapping (Video S1,
The ferning patterns of the biofilms were large (visible without microscopy on the order of centimeters) with most of the patterns consisting of orthogonal branches, and the ferns were reproducible in coverage and complexity score. The ferning patterns that were formed by the biofilm had the same morphology as ferning patterns of saliva, cervical mucus, E. coli, salt-gel, or salt-protein [44, 47, 49, 50]. From reports in the literature and the results in this work, the orthogonal or oblique branching seemed to be the most common type of ferning with the examples of branches with acute angles being rare [47]. The cause of the change in the morphology of the fern in biofilms from 90° angles to acute angles is not immediately clear. However, other studies have reported that the gelatin-to-salt ratio was the key factor controlling the ferning morphology of salt-gelatin mixtures [47]. Therefore, the samples with higher rheological properties (glycerol and glucose samples), which arguably has a higher amount of EPS, may have produced branching with acute angles due to the increased EPS-to-salt ratio. Thus, the acute-angle morphology dominated when the biofilms had larger rheological values, indicating higher EPS-to-salt ratio, while orthogonal-branching morphology dominated at intermediate ratios with no ferning at extremely high or low values of the EPS-to-salt ratio.
Analyzing the pre-desiccation biofilm that was grown in 5% NaCl under polarized filters showed the entire sample sample lit with birefringent strands (Figure 6). The Pf bacteriophages produced by P. aeruginosa are known to self-assemble into liquid crystals that exhibit birefringence [64]. The birefringent strands (black arrows) are 50–100 μm in length, and they are evenly distributed throughout the sample (Figure 6a and b). A magnified view of the strands revealed that each strand was a bundle of smaller strands that were surrounded by cell clusters (yellow arrows) and biofilm (Figure 6c). The bacteriophages are about 6 μm in length, so the bundle was likely composed of hundreds of individual Pf phages that were assembled into one strand [64]. These birefringent strand clusters did not exist in samples lacking biofilm. A plate with 10% NaCl LB medium formed sheets of salt crystals, no visible biofilm, and no birefringent strands (Figure S6,
Polarized microscopic images of inoculated samples of PAO1 in modified LB medium with a concentration of 5% NaCl. (a and b) Birefringent fragments exist throughout the liquid medium. (c) The birefringent thread-like fragments (black arrows) of about 50–100 μm in length were dispersed within medium that was full of bacterial clusters (yellow arrows).
The 5% NaCl sample that was previously analyzed (Figure 6) was desiccated (Figure 7a) and examined with and without a polarized filter (Figure 7b–k). The red lines outlined the specific regions of the fern under the microscope. The region outlined in the circle (Figure 7b–e) was the square crystalline structure on the plate. Without a filter this region showed a cubic structure with a length of 4–5 mm per side with a nucleation point in the center and thin diagonal lines running through it with cavernous voids coming from the sides of the structure (Figure 7b). The center of the ferning structure showed cuboid lattice-like patterns of growth emerging from the seed point (Figure 7e). Similarly, the previous study of the ferning pattern in a gelatin-salt mix revealed interlocking salt blocks with a length of 10–30 μm on a side that formed the backbone of the ferning structure [47].
Desiccated biofilm that was grown in LB medium with 5% NaCl. (a) Ferning pattern from the bottom of the petri dish. The regions that are outlined in red were inspected under the microscope. (b–e) The cubic piece at the top right corner of the plate (red circle): (b) seen under normal light; (c and d) seen through a polarized filter at different magnifications; (e) focused on the seed point of crystallization. (f and k) The ferning region of the plate (red square): (f–h) different regions of the fern under normal light at different magnifications; (i and j) seen through a polarized filter at different magnifications; (k) viewed at a different angle of polarization.
With a polarized filter, bright birefringent regions lit throughout the square (Figure 7c). A closer look at the center of the square revealed two forms of birefringent structures, large red and gold star formations (~0.5 mm in diameter) and red and gold strands that were about 50 μm in diameter (Figure 7d). In contrast to the birefringent strands that were scattered throughout the sample (black arrows), the birefringent stars (white arrows) were only in the crystallized ferns. Each birefringent strand that was visible in Figure 7c was a bundle of even smaller birefringent strands (Figure 6c). Therefore, the formation of the birefringent star demonstrated that having an even higher order of self-assembly during desiccation was possible such that the bundled strands further merged into a star formation. The alternating red and gold coloring indicated that the strands with matching orientations cluster together, but they must not have formed the entire cluster, as no star formation existed with only one color.
From the linear ferning section that was outlined by the red square (Figure 7f–k), the branches appeared to be about 1 mm in width with a distinct centerline running through each branch (Figure 7f). A magnified view of one of these branches revealed latticed or layered networks emanating from this central line (blue arrows) and cavities (pink arrows) that were present throughout the structure (Figure 7g). Some of the cavities were large, tunneling deep into the ferning structure (Figure 7h). Under polarized light, the branch was shown to have dozens of the star-shaped red and gold birefringent bundles (Figure 7i). Changing the angle of the polarized filter changed the color of the birefringent region from red and gold to gold and green (Figure 7k). The star-shaped birefringent clusters only existed within the crystal regions of the fern pattern, while the strands were scattered throughout the plate regardless of the ferning pattern (Figure 7j). This localization of the morphologies implied that the birefringent strands were produced within the biofilm; thus, they could be found throughout the material, while the formations of the birefringent stars were created as a result of crystallization, so they were only found within the crystalline regions. Clusters of bacterial cells appeared to be entrapped within the crystallized fern (yellow arrows), especially around the extremities of the ferning structure (Figure 7c and j). Similarly entrapped bacterial clusters were capable of reanimation at least a week after desiccation within the ferning structure [50]. Therefore, clusters of P. aeruginosa that were seen in Figure 7c and j may be in a suspended animation state as well, though this hypothesis was not tested during this study.
In environments that contained high viscosity (glycerol), high osmolarity (glycerol, NaCl), and high concentrations of simple carbon (glucose), the elasticity and the yield stress of the biofilm increased. Silver nitrate had an inhibiting effect on the biofilm formation, but only at concentrations that were greater than 0.1 mM. Similarly, concentrations of glycerol greater than 10% completely inhibited biofilm growth. However, the complex carbon structure of sucrose meant that it could not be utilized as an additional carbon source by PAO1 in the same way that glucose was utilized. Therefore, sucrose did not change the rheological properties of the biofilm. So, P. aeruginosa developed stronger biofilm under nutrient-rich conditions, certain levels of osmotic stress, and certain levels of diffusion limitation. However, it would not develop biofilm when the osmotic stress or diffusion limitation exceeded an inhibition amount or when an antimicrobial agent exceeded its inhibition concentration.
While the rheological properties of biofilm revealed information about the strength of the biofilm, the morphology of the ferning pattern best described the interactions between the electrolytes and the EPS in the biofilm. Typically, the biofilm had ferning coverage of about 50% and a ferning complexity score of 5. The ferning complexity increased with the strength of the biofilm (high complex modulus and yield stress), as stronger biofilm increased diffusion limitation that was experienced by the solutes within the matrix. The coverage and complexity score both dropped to zero when no biofilm formed, so the macromolecule-to-salt ratio was too low for ferning to occur, as with high concentrations of silver nitrate and glycerol. Many of the analysis methods of biofluid ferning patterns were qualitative and subjective, which is currently problematic considering its use as an indicator of certain medical symptoms. The image analysis and ferning classification method that was presented here could easily be applied to the other fields to give more quantitative values to the analysis of ferning biofluids.
The birefringence that was produced by liquid crystals within the samples of biofilm had two different morphologies, bundled strands that were about 50 μm in length in hydrated biofilm and star-shaped bundle of strands that were almost ten times larger inside the crystalline region of the ferning pattern. So, in addition to the self-assembly of the phages to strands inside the biofilm, a more complex assembly took place during crystallization in the biofilm that produced this tertiary structure. During the ferning process, clusters of bacteria became entrapped within the crystalline phase. Other researchers have found that these entrapped bacteria are in suspended animation state and that they could be brought back to life upon rehydration. If PAO1 can also reanimate, then ferning is yet another mechanism that P. aeruginosa could utilize to survive extreme conditions, similar to how liquid crystals formed by phages enhanced the resistance and persistence of P. aeruginosa.
The authors would like to thank Dr. Skip Rochefort for consulting on the rheology tests and Kristin Marshall for her help with the image conversion work. Additionally, the authors would like to thank Marisa Thierheimer, Curran Gahan, and Dalton Myas for helping with experimental preparation.
We do not have any conflict of interests to declare.
This work would not be possible without funding from Medical Research Foundation of Oregon.
The supplemental documents for this section may be found at:
Edited by Jan Oxholm Gordeladze, ISBN 978-953-51-3020-8, Print ISBN 978-953-51-3019-2, 336 pages,
\nPublisher: IntechOpen
\nChapters published March 22, 2017 under CC BY 3.0 license
\nDOI: 10.5772/61430
\nEdited Volume
This book serves as a comprehensive survey of the impact of vitamin K2 on cellular functions and organ systems, indicating that vitamin K2 plays an important role in the differentiation/preservation of various cell phenotypes and as a stimulator and/or mediator of interorgan cross talk. Vitamin K2 binds to the transcription factor SXR/PXR, thus acting like a hormone (very much in the same manner as vitamin A and vitamin D). Therefore, vitamin K2 affects a multitude of organ systems, and it is reckoned to be one positive factor in bringing about "longevity" to the human body, e.g., supporting the functions/health of different organ systems, as well as correcting the functioning or even "curing" ailments striking several organs in our body.
\\n\\nChapter 1 Introductory Chapter: Vitamin K2 by Jan Oxholm Gordeladze
\\n\\nChapter 2 Vitamin K, SXR, and GGCX by Kotaro Azuma and Satoshi Inoue
\\n\\nChapter 3 Vitamin K2 Rich Food Products by Muhammad Yasin, Masood Sadiq Butt and Aurang Zeb
\\n\\nChapter 4 Menaquinones, Bacteria, and Foods: Vitamin K2 in the Diet by Barbara Walther and Magali Chollet
\\n\\nChapter 5 The Impact of Vitamin K2 on Energy Metabolism by Mona Møller, Serena Tonstad, Tone Bathen and Jan Oxholm Gordeladze
\\n\\nChapter 6 Vitamin K2 and Bone Health by Niels Erik Frandsen and Jan Oxholm Gordeladze
\\n\\nChapter 7 Vitamin K2 and its Impact on Tooth Epigenetics by Jan Oxholm Gordeladze, Maria A. Landin, Gaute Floer Johnsen, Håvard Jostein Haugen and Harald Osmundsen
\\n\\nChapter 8 Anti-Inflammatory Actions of Vitamin K by Stephen J. Hodges, Andrew A. Pitsillides, Lars M. Ytrebø and Robin Soper
\\n\\nChapter 9 Vitamin K2: Implications for Cardiovascular Health in the Context of Plant-Based Diets, with Applications for Prostate Health by Michael S. Donaldson
\\n\\nChapter 11 Vitamin K2 Facilitating Inter-Organ Cross-Talk by Jan O. Gordeladze, Håvard J. Haugen, Gaute Floer Johnsen and Mona Møller
\\n\\nChapter 13 Medicinal Chemistry of Vitamin K Derivatives and Metabolites by Shinya Fujii and Hiroyuki Kagechika
\\n"}]'},components:[{type:"htmlEditorComponent",content:'This book serves as a comprehensive survey of the impact of vitamin K2 on cellular functions and organ systems, indicating that vitamin K2 plays an important role in the differentiation/preservation of various cell phenotypes and as a stimulator and/or mediator of interorgan cross talk. Vitamin K2 binds to the transcription factor SXR/PXR, thus acting like a hormone (very much in the same manner as vitamin A and vitamin D). Therefore, vitamin K2 affects a multitude of organ systems, and it is reckoned to be one positive factor in bringing about "longevity" to the human body, e.g., supporting the functions/health of different organ systems, as well as correcting the functioning or even "curing" ailments striking several organs in our body.
\n\nChapter 1 Introductory Chapter: Vitamin K2 by Jan Oxholm Gordeladze
\n\nChapter 2 Vitamin K, SXR, and GGCX by Kotaro Azuma and Satoshi Inoue
\n\nChapter 3 Vitamin K2 Rich Food Products by Muhammad Yasin, Masood Sadiq Butt and Aurang Zeb
\n\nChapter 4 Menaquinones, Bacteria, and Foods: Vitamin K2 in the Diet by Barbara Walther and Magali Chollet
\n\nChapter 5 The Impact of Vitamin K2 on Energy Metabolism by Mona Møller, Serena Tonstad, Tone Bathen and Jan Oxholm Gordeladze
\n\nChapter 6 Vitamin K2 and Bone Health by Niels Erik Frandsen and Jan Oxholm Gordeladze
\n\nChapter 7 Vitamin K2 and its Impact on Tooth Epigenetics by Jan Oxholm Gordeladze, Maria A. Landin, Gaute Floer Johnsen, Håvard Jostein Haugen and Harald Osmundsen
\n\nChapter 8 Anti-Inflammatory Actions of Vitamin K by Stephen J. Hodges, Andrew A. Pitsillides, Lars M. Ytrebø and Robin Soper
\n\nChapter 9 Vitamin K2: Implications for Cardiovascular Health in the Context of Plant-Based Diets, with Applications for Prostate Health by Michael S. Donaldson
\n\nChapter 11 Vitamin K2 Facilitating Inter-Organ Cross-Talk by Jan O. Gordeladze, Håvard J. Haugen, Gaute Floer Johnsen and Mona Møller
\n\nChapter 13 Medicinal Chemistry of Vitamin K Derivatives and Metabolites by Shinya Fujii and Hiroyuki Kagechika
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