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Vanilla Bean: History, Origins and Uses

Vanilla Bean come from the Vanilla Orchid Plant, it is a kind of rare natural plant species. The best producing area is Africa island of Madagascar, One of the easier to grow indoor orchids. They look like green beans when they’re ripe and after picking need to be dried and fermented for their rich flavour to develop. It’s a fairly complicated and lengthy process, which is why the price is so high. Vanilla beans only grow in tropical climates. Vanilla bean has elegant full-bodied milk sweet fragrance, widely used in high-grade food processing, such as: ice cream, cake, milk, sweet drinks, tea, wine, meat processing and puffed food. Vanilla bean is the second most expensive spice then saffron of the world. (1 ServoLux, 2007)
History Vanilla plan folia are indigenous to Mexico and may have been used up to 1000 years ago by the Totonac tribe as a flavoring. The Totonacas still grow vines with almost religious devotion because to them it was the gift of the gods. It is not uncommon to have a few vines growing around their houses. These are watered every day as if they were the Tontonacas most valuable possession. The vanilla beans were used as a tribute to the Emperor of the Aztecs.
In 1518 the Spanish Conquistador, Herman Cortez, met with Emperor Montezuma while seeking treasures of the New World. He observed that the Emperor enjoyed a royal beverage of vanilla scented chocolate; Cortez was so impressed by this regal drink that when he returned to Europe, he took bags of cocoa and vanilla along with the gold, silver and jewels of Montezuma’s fallen empire. Within half a century, Spanish factories were preparing vanilla-flavored chocolate. For quite some time the Europeans continued to use vanilla only in combination with the cocoa bean.
By 1602 vanilla began to be used as a flavoring on its own – the suggestion of Queen Elizabeth’s apothecary, Hugh Morgan. From then vanilla soared in popularity and became more famous than chocolate or any other flavor known before or since. For more than 300 years after its discovery by Cortez, vanilla was produced only in its native Mexico.
Plants were tried in many countries but the orchids never bore fruit. The mystery was not solved until 1836 when a Belgian named Charles Morran found that common insects could not pollinate the orchid. He observed that a tiny bee, which is found only in the Vanilla districts of Mexico, is uniquely equipped to pollinate the flowers. The bee did not survive outside Mexico and so Morren developed a method of hand-pollinating the Vanilla blossoms.
Soon after this discovery, the French started to cultivate Vanilla on many of their islands in the Indian Ocean, East and West Indies and Oceania; the Dutch planted it in Indonesia; and the British took it to southern India. Eventually the French took Vanilla to Reunion, an island off Madagascar. There a former slave named Edmond Albinus perfected a quick and simple method of hand-pollination which is still used to this day. (2 ServoLux, 2007)
Geography Traditionally, Vanilla requires warm and moist conditions with well distributed rainfall of 150 to 300 cm with a temperature range of 25 to 32oC. It comes up well from sea level to around 1500M above mean sea level. The crop requires more than 50% shade and thrives best under filtered light. Vanilla comes up well in loose and friable soil with very high organic matter content and of loamy texture. It prefers land with gentle slope and well-drained soils. (3 ORCHIDSASIA, 2009)
Originating in Central America, the main distribution about, Mexico, Madagascar, and Indonesia tropical Marine areas, Mauritius, Sri Lanka, and India also has a small amount of cultivation, China Fujian, Guangdong.

Madagascar – Madagascar is the biggest vanilla bean producer. Vanilla bean is growth in the island of Madagascar, Comoros and La Reunion. This area damp, belong to the high temperature, the rain is abundant, and the soil moisture, these conditions conducive to vanilla growth.
Indonesia – The second kind of growth in Indonesia, this area belongs to the tropical climate, the altitude of 1500 meters, the temperature in the 21 – 32, air humidity is in commonly 75%, suitable for vanilla growth.
China – In China’s Guangdong, Fujian have a small amount of vanilla growth, because in this area the region perennial temperature wet, rainwater enough, winter wet rain. Soil is weak acid suitable for vanilla growth.
Mexico – Mexico is hot and humid with climate; annual rainfall of 1500-3500 mm.Temperature is 20-30 between, Mexico region’s soil loose, good drainage, suitable for vanilla growth.
Propagation, Planting, Harvesting The crop is usually established by planting in situ shoot cuttings each, preferably having 8 to 10 inters nodes as these flower earlier than the shorter cuttings. However, the cuttings with less than five to six inter nodes and 60 cm in length should not be used directly for planting. Such shorter cuttings properly rooted in the nursery, establish well in the field, compared to the stem-cuttings. Micro- propagated plants can also be used for planting. Vanilla is propagated mainly by shoot cuttings or rooted cuttings. Strong, healthy and actively growing vines are selected, cut into pieces of one meter long with three or four leaves removed from the bottom. The cuttings are kept in a shady place for one week. Alternatively, 3-4 nodded rooted cuttings are also used for planting.
For optimum growth of these plants, a controlled environment is created by establishing suitable green-house/ shade net house which provides the appropriate amount of light, temperature and humidity which are essential for commercial production of vanilla. High-density polyethylene net providing 50-60% shade can be supported with stone pillars of 12? height to provide the required shade. Micro-sprinklers with both irrigation and misting/ fogging facility need to be installed in the shade house, which will ensure the irrigation as well as humidity requirements.
Vanilla is planted in a medium rich in organic matter. Decomposed organic manure is filled in the trenches made at a spacing of 8?. In these trenches, support pillars of 7? long will be placed at a spacing of 6?, and two cuttings each, will be planted around one support pole. The plant density per acre thus works out to 2400.
The main source of nutrients for the crop is from organic sources viz., decomposed leaf mould or dry. A thick layer of organic debris also helps to retain enough moisture and gives a loose soil structure for the roots to spread. Hence, it is important that easily decomposable organic matter is applied around the plant base at least 3-4 times in a year.
The flowering commences from the 3rd year after planting, during January – February months. After pollination and fertilization, the beans develop very quickly and obtain full size in about 5-6 weeks. The beans are harvested when the distal end turns pale yellow in color. The aroma and flavor develops only after the curing process. The aroma and flavor develops only after the curing process. The different stages of curing include Killing, Sweating Drying and Conditioning. (5 ORCHIDSASIA, 2009)
Types of vanilla Madagascar Bourbon (plan) is the most common been used in extracts. Bourbon beans from Madagascar and the Comoros are described as having a creamy, hay-like, and sweet aroma, with strong vanillin overtones. The bourbon-Madagascar bean is sweet, long and slender and has a rich, full flavor and oily skin. (6 ServoLux, 2007)
Mexican vanilla beans, also plan, are very similar to Madagascar beans though they have a mellower, smooth quality and a spicy, woody fragrance. Dark chocolate, dairy desserts, beverages, poultry and meat are complemented by Mexican vanilla. The Mexican bean has a smooth, rich flavor and spicy aroma.
Madagascar and Mexican vanillas both provide the familiar natural vanillin flavor that we associate with vanilla ice cream and other vanilla-flavored desserts and beverages. They are the gold standard of the vanilla market. (7 ServoLux, 2007)
Tahitian vanilla beans (vanilla tahitensis) originate from plan stock that was taken to Tahiti, where it mutated in the wild. It is now classified as a separate species (Vanilla tahitensis) as it is considerably different in appearance and flavor from Plan vanilla. The beans are often described as smelling like licorice, cherries, prunes, or red wine. Tahitian beans offer a more floral fruity flavor most suitable in savory and fruit dishes. The Tahitian bean is thick, dark and has a thinner skin and fewer seeds compared to the other beans; plus, it has a higher oil and water content. (8 ServoLux, 2007)
Culture In the history, the Spanish people think vanilla is a stimulant; the Indians call it god’s fruit. The Aztecs use it as a currency. In the past eight hundred years vanilla this magic little pod life Mexico and Madagascar’s economy; Develop the Indian trade; Even consolidate strengthen the foundation of Madagascar, on the world map occupies a position; Vanilla is a kind of spices, is widely used in the world snacks and food. More than 19% of artificial spices contain vanilla composition.
(9 Patricia Rain, 2004)
Culinary use Vanilla Bean is used spices in the world, people often use it in food for flavoring, and it is Luscious, warm, sexy and exotic. And it can be used in cake, pudding, milk, ice cream, coffee and tea. Vanilla Bean gives better flavor to these foods.
(10 Greenvanillastore 2012)
Non-culinary use Vanilla can make into vanilla fragrance product, such as Candle, Body wash, Shampoo ,body perfume it has a lot of health care effect, can remove banned anxiety, relieve pressure, relaxation nerve, help sleep. Vanilla can also make soap, can have the effect of hairdressing, and maintains the skin to add on the fragrance. (11 vanillabazaar, 2012)
Medical use Vanilla contains 150-170 kinds of aroma components and 17 kinds of human body essential amino acids, which is can be tonifying kidney, appetizers, tonifying spleen, medical effect, it is a kind of natural nourishing medicine, is known as “the king of natural spices”( 12unknow,2010)
Change agents Totonca – Totonca is the first to discover the vanilla beans in the world, vanilla was a sacred herb that they incorporated into all levels of their lives. They use the Vanilla Bean to made garlands worn around the neck as a protective character, in order to prevent a variety of diseases. (13 the vanilla company, 2012)
Herman Cortez – In 1518 the Spanish Conquistador, Herman Cortez, met with Emperor Montezuma while seeking treasures of the New World. He observed that the emperor like royal beverage vanilla fragrance chocolate, Cortez left a very deep impression, when he returned to Europe; he took bags of cocoa and vanilla Montezuma Empire. For quite some time the Europeans continued to use vanilla only in combination with the cocoa bean. (14 ServoLux, 2007)
Hugh Morgan – he is the Queen Elizabeth’s apothecary, in 1602 he suggestion that vanilla can be used as a flavoring. From then vanilla soared in popularity and became more famous than chocolate or any other flavor known before or since. (15 ServoLux, 2007)
Carl Lai ham – in 1898, the United States north Carolina new Berne young pharmacist Carl。Lai ham invented by a kola nut and vanilla seeds mix drink at first, it was used in the treatment of digestive, but when he mixed soda water in this drink, he found a delicious refreshing drinks then it is named vanilla cola, it was called Brads drinks. (16 unknow, 2010)
Innovation Natural Vanilla Flavor: a mix of pure vanilla and other natural substances other than the vanilla bean. It usually is made with a glycerin or a propylene glycol base. (17 ServoLux, 2007)
Vanilla-vanillin: a mix of pure vanilla extract and synthetic substances, most commonly vanillin. (18 ServoLux, 2007)
Vanilla powder: a mixture of ground vanilla beans and vanilla oleoresin combined with carbohydrate carriers and flow agents. It is not that kind of white chemical food, but the same color pod black or brown black powder, is a kind of natural vanilla bean into flour products. (17 vanillabazaar, 2012)
Vanilla cola: Carl Lai ham invented by a kola nut and vanilla seed mix drink at first, it was used in the treatment of digestive, but when he soda water with the mixed, he found a delicious refreshing drinks in was named vanilla cola before, it was called Brads drinks. (18 unknow, 2010)
Vanilla sugar: a product made from vanilla powder and sugar, it can be use for bake, add in some drink.
Vanilla extracts: it is made with vanilla beans and it is no sugar, offer a fresh clean flavor to cuisine. (19 ServoLux, 2007)
Nutrition Per 100 g Vanilla bean About 130 other compounds have been identified in vanilla extract. Vanillas also contain water (35%), sugars (25 %), fat (15 %), cellulose (15% to 30 %t) and minerals (6 %). (20 unknow, 2010)
Resources Human resources the main distribution about, Mexico, Madagascar, and Indonesia tropical Marine areas, Mauritius, Sri Lanka, and India also has a small amount of cultivation, China Fujian, Guangdong. In these countries (apart Mexico), Artificial pollination by hand is the rule for fruit setting. 85 to 100 per cent success is obtained by hand pollination. And the curing process needs more manual
Natural resources Vanilla requires warm and moist conditions with well distributed rainfall of 150 to 300 cm with a temperature range of 25 to 32oC. And it requires more than 50% shade. Vanilla comes up well in loose and friable soil with very high organic matter content and of loamy texture. It prefers land with gentle slope and well-drained soils.
Mechanical resources Vanilla bean processing need a curing process, in this process need use the oven, set oven 45-50 to curing it.
Future trends The vanilla bean growth in the warm and moist areas, and I believe that with progress of the era, vanilla bean maybe can planted in indoor, and harvest in different season.
Contemporary uses and applications In the bakery, such as vanilla slice.
In the ice cream, such as vanilla ice cream.
In Sweet dessert, such as vanilla putting.
From the procession of such as vanilla milk, vanilla coke, vanilla powder, vanilla syrup.

Zeta Potential of Liposome Production

Empty and drug loaded liposomes were prepared with different lipid compositions and by different methods. These liposomes were analysed as this types of liposome have particle size in the range needed for aerosol delivery and delivery of drug to the deep lung deposition. In order to interpret which parameters are significant for the preparation of liposomes that can be used for such applications, eight different lipid membrane compositions were studied. The drug molecule used was tobramycin, as there is therapeutic rationale for its use in cystic fibrosis, but also because of its complex structure, hydrophilic nature and it is also believed that it is difficult drug to encapsulate into liposome vesicles. Thus results and findings of this study can be used as a basis for the formulation of liposomes intended for delivery to the lungs by nebulisation.
Liposome Surface Charge The zeta-potential of different liposome formulation without tobramycin were obtained, values (Table 6) indicates difference in charges based on different lipids used in the formulation.

As DPPC is a neutral lipid, it produces liposomes with no surface charge on the liposomes as seen in formulation T1 and T2. The addition of cholesterol to the liposome formulation caused no change in surface charge. Values with DSPE-PEG lipids were found close to zero, indicating an overall neutral charge for these liposomes (zeta potential for T3 and T4 liposomes formulation was measured to be -6.46±0.42 mV and -7.24±0.14 mV respectively). As expected for negatively charged (DPPG-Na) liposomes, the zeta potential was more negative (-66.43± 2.95 mV), which indicates lipid membranes are negatively charged. It is readily observed that the zeta-potential values of the negatively charged liposome, is decreased by presence of DSPE-PEG and cholesterol such as in formulation T6, T7 and T8. DPPG represents the main component of bacterial membranes and is a minor component of lung surfactant. Unlike DPPC, head groups of DPPG lipid molecules have a net negative charge (anionic) (Ianoul, et al., 2007) and higher membrane rigidity (Kinman, et al., 2006). It is therefore expected that since the tobramycin is a positively charged, their interaction with DPPG-Na phospholipid will be stronger.
Size of Liposomes The particle size distributions of the different formulation with and without tobramycin obtained from Mastersizer, presented in Tables 7 and 8; are found to be unimodal and range from 1.0 to 4.0 µm.

Liposome formulations with or without cholesterol did not show any great differences in the size characteristics. With the addition of tobramycin, it is observed that size of the liposome decreases this is due to the fact that tobramycin has hydrophilic nature and positive charge. It can be seen that after including DPPG-Na, size of the liposomes decreases as it is negatively charged and tobramycin is positively charged so due to electrostatic interaction, its size is reduced or decreased aggregation. It can be seen from the results, liposome of tobramycin are within the range which are required for an optimal deep lung deposition.
Size distributions of the various types of liposome after sonication for 10 min are presented in tables 9 and 10. Effect of various lipids and cholesterol is seen on the size distribution of different liposome formulation with or without tobramycin.

All liposomes formulations are within range from 90.0 to 250 nm (i.e. small unilamellar liposomes or small multilamellar liposomes). In most liposome formulation, the polydispersity indexes measured are low indicating that the vesicles are monodisperse. Addition of cholesterol in the lipid membranes of liposomes resulted in increased vesicle size as previously reported (Zaru, et al., 2007). Incorporation of tobramycin to the liposome formulation (Table 10) increased the size of liposome in comparison to empty liposomes prepared under identical conditions. These liposomes incorporate tobramycin in an aqueous core as it is hydrophilic drug (Ramana, et al., 2010). The bulky size of tobramycin may responsible for the increased size of the tobramycin loaded liposomes compared to empty liposomes.
Selected formulations were directed into the Sympatec particle sizer in order to determine the size of the aerolised particles as presented in Table 11 and 12. It can be observed from both tables that droplet size is in range 3-5 µm as required for the pulmonary delivery.
From table 11, it can be interpreted that addition of cholesterol to the empty liposomes had very little effect on droplet size. DSPE-PEG increases the size of the droplet size in both empty as well as drug loaded liposome while DPPG-Na decreases the size of the droplet size. Droplet sizes are in the respirable range. There is no major difference in droplet size, in comparison to with or without tobramycin. Droplet size is a function of the nebuliser rather than the liposomes formulations.
Liposome Encapsulation Efficiency
The liposome encapsulation efficiency % measured for encapsulation of tobramycin liposomes with different lipid composition and different methods used, is presented in Figure 7. Formulations (T1 – T9) are prepared by the proliposome method and without sonicated samples were used. Formulations TFM1 and TFM2 are prepared by the thin film method. As seen, tobramycin liposome encapsulation is within range from 5 to 20% for proliposome method. Lipid composition is a very important determinant of Tobramycin encapsulation. The amount of tobramycin encapsulated in liposomes increased significantly when liposomes are formed from DSPE-PEG and DPPG-Na lipids compared to DPPC based liposomes. Inclusion of cholesterol in the lipid bilayers, results in a decrease of tobramycin encapsulation efficiency % in formulations T2, T4and T6 (due to displacement of drug from the bilayers by cholesterol). Usually, the charged liposomes containing DPPG-Na lipid express considerably higher encapsulation ability (Liposome Encapsulation Efficiency % is 15 and 10% for T5 and T7 formulations, respectively) compared to the equivalent uncharged liposomes.
In the formulation T9, the effect of chitosan coating on tobramycin loaded liposomes was observed as liposome encapsulation efficiency. As previously reported, when liposomes are prepared with chitosan solution, it forms chitosan coated liposomes as polymer adheres to the liposomal surface (Takeuchi, et al, 1996 and Takeuchi, et al., 2003). From the results obtained from this study, it is evident that electrostatic interactions are involved in the liposome coating with chitosan solution. This can be proven by the fact that negative charge (DPPG-Na) of liposome is modified as they are coated with polymer; while non-charged liposome, were not significantly modified, therefore they have very low coating efficiency. In case of neutral liposomes, they can be coated with chitosan solution but they will have lower efficiency in comparison to negatively charged ones (Mobed, et al., 1992). It has been previously noted that chitosan coating on neutral liposome involves hydrogen bonding between the phospholipid head groups and the polysaccharides (Perugini, et al., 2000). In respect to tobramycin encapsulation efficiency, an increase in encapsulation efficiency was observed in chitosan coated liposomes (Figure 7) in comparison to non-coated liposome formulations.
Thin film hydration is a simple technique for the preparation of liposomes, but its major disadvantage is poor encapsulation efficiency of hydrophilic drugs. Furthermore, encapsulation of the drug decreases with the reduction in size of liposome (Sharma and Sharma, 1997). Therefore, it indicates from the result that encapsulation efficiency (Figure 7) of liposomes prepared by thin film hydration method (TFM 1 AND TFM 2) is low compared to those formulations prepared by the proliposome method.
Figure 8, represents surface association of tobramycin solution on to the empty liposomes. Drug loading technique like adsorption on to preformed empty liposome is widely described. It is difficult to encapsulate the drug with small size particles; therefore, the drug is adsorbed onto the surface of the empty liposomes rather than encapsulation (Almeida and Souto, 2007). Adsorption % from the graph (1 – 1.5 %) is negligible in comparison to encapsulation of the drug. Therefore, encapsulation is a better method for drug entrapment onto the liposomes.
Twin Stage Impinger (TSI) deposition of nebulised formulations Figure 9, represents drug deposition after the sonicated liposome formulations were subjected to nebulisation. Tobramycin liposomal formulation is more efficiently delivered to stage 2 (cut-off diameter of particles delivered to stage 2 is less than 6.4 µm) of TSI than the tobramycin solution. In all the liposome formulations, nebulisation efficiency was higher than 65 percent of initial materials contained in the nebuliser and this is not practically affected by lipid composition present in different liposome formulation. It is interested to note that the presence of lipids distinctly enhances the deposition in stage 2 of the twin stage impinger (TSI). As shown in Figure 9, the presence of lipid coat around tobramycin particles allowed decrease in deposition in the nebuliser device, while there is increase in deposition in stage 2. The stage 2 deposition of uncoated tobramycin is around 49%, which is increased by up to 66-71% for the lipid coated formulations. Therefore it is extremely helpful for the patients in terms of deep lung penetration and drug targeting efficiency. As expected, there is negligible amount of tobramycin observed in stage 1 of TSI. There is no effect after addition of cholesterol to the lipid membrane, on nebulisation efficiency. This may be due to the hydrophilic nature of the drug and because cholesterol is forming leaky lipid membranes.
The deposition of the non-sonicated liposome formulation after nebulisation is shown in Figure 10. The aerosolisation and deposition performance of the tobramycin liposome were analysed at two stages of twin stage impinger. This finding has resulted in reduction of deposition in nebuliser device, whereas it increased deposition in the lower stage of twin stage impinger. From the Figure 10, it can be said that after inclusion of DSPE-PEG to the formulation, deposition in lower stage is increased from 63 – 80% (liposome formulation T5 and T6) to 96% (liposome formulation T7 and T8). There was no tobramycin detected in upper stage of TSI. After comparison of the nebulisation efficiency of the sonicated and non-sonicated, it can be observed that deposition in lower stage is more with non-sonicated samples but this experiment was performed only once.
The size of the nebulised liposomes was measured before and after nebulisation at different stages of TSI as shown in Table 13 and 14. As seen in Table 13, size of the sonicated liposomes is not changed on passage through nebuliser for the formulation T5 and T6. But there is reduction in size after nebulisation for the formulation T7 and T8, this may be due to the fact that liposomes were disrupted and further loss of the entrapped drug, which is hydrophilic in nature (Elhissi, et al., 2007).

From Table 14, it can be observed that size is decreased in nebuliser compared to before nebulisation for non-sonicated liposomes but there is very little difference in particle size present in nebuliser and stage 2 for the formulations T5 and T6. For the formulation T7 and T8, it can be seen that size of the particle is increased in stage 2 as compared to nebuliser and stage 1. It is to be expected that liposomes have aggregated in the lower stage impinger after they were delivered from the air jet nebulizer and this may be due to close proximity of the liposomes or disruption of the liposomes which led to aggregation of liposome fragments (Elhissi, et al., 2010).
Conclusion From this study it can be concluded that depending on the composition of lipid and method of preparation of lipid, liposomes have advantage of acting as carrier for delivering tobramycin to the lung. Also based on coating of liposome with chitosan, various mixtures of phospholipid and cholesterol used can improve delivery of tobramycin to deep lung deposition. The size of all the liposome formulation was within the range which is suitable for lung deposition. Drug loading may be increased with the use of chitosan solution. From this study, it demonstrated the effect of negatively charged lipid (DPPG-Na) with the positively charged tobramycin that encapsulation efficiency and nebulisation efficiency can be increased in comparison to non-charged ones. Therefore, liposomal tobramycin is good formulation in comparison to tobramycin solution.
Future Work From the results of this study, it has been demonstrated that chitosan coated liposomes offers advantage over non-coated ones. Liposome prepared by chitosan solution has more encapsulation efficiency. But limitation from this study is that it was performed with only one lipid compoistion. So liposome of different lipid compositions can be formulated with chitosan solution and characterised for its liposome size, drug loading and twin stage impinger for further studies. Even negatively charged lipid is a promising lipid for further studies with positively charged tobramycin. The twin stage impinger studies can be done with the use of a different nebuliser, particularly a vibrating-mesh nebuliser would be useful.

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