K

Membrane Filtration where it is used to raise the temperature of the incoming raw gel to about 50—60 °C while its own temperature decreases to about 35—45 °C. A final heat exchanger cools the leaving gel to the desired temperature with chilled glycol or water. Bacteriologically, the kill efficiency of this HTST process is inherently no better than properly conducted batch processing. However, HTST is capable of handling larger volumes and functions in an automated fashion. HTST can more efficiently reach the high temperatures needed to handle micrococci. However, the caveats inherent in the heavy bacterial loads of the WLE process still apply.

Recently, a modification of HTST has proven successful in the dairy industry. This process is called ultrapasteurization (UHT). In this case, the product stream is not only sealed but is pressurized such that temperatures in excess of 100 °C can be attained. To our knowledge this promising process has not been employed to date on a large scale in the aloe industry.

In the pharmaceutical industry sterilization of liquids by filtration through submicron membrane filters has been common for decades, and this process is utilized to a lesser degree by the food and beverage industry. This process (Figure 8.6, Process L) has not been widely adopted by the aloe industry, although some small scale production of specialty products employs membrane sterilization. This process is industrially impractical for high quality raw aloe gel because the extreme pseudoplasticity results in unacceptable back pressures. If pseudoplasticity is broken by degradation of the native polysaccharide with enzymes, then aloe liquid can be membrane sterilized, although it is no longer a physical gel. On a laboratory scale, membrane filtration is very commonly used to sterilize aloe. For purposes of HPLC analysis a 25 mm, 0.2 ^m, filter can be used to treat the native gel from 1—2 ml of aloe yielding the 100—200 ^l of filtrate required for HPLC analysis. However, it is physically very difficult to manually prepare even 10 ml of high quality aloe gel by direct passage through a 0.2 ^m filter. The gel must first be 'broken' by passage through multiple filters of decreasing porosity, usually first a glass fiber filter, followed by a 20 ^m filter, then 5 ^m, then 1.2 ^m, prior to final 0.2 ^m filtration. Investigators who have not worked with native gel do not appreciate the pseudoplasticity of native gel since commercial aloe lacks this property.

The product

The processes described above yield a very slightly yellow to almost water white liquid. In the case of gel of the highest quality, the material is significantly pseudoplastic. In our studies of the chemistry and biology of Aloe barbadensis gel (Strickland and Pelley, Chapter 12), this material, lyophilized without addition of preservatives or further processing and is termed ARF Process B material. This material, usually with the addition of preservatives, is marketed as '1:1 Aloe Vera Gel' (IASC, 2001). Ideally when mesophyll contamination is low, pasteurization does not affect the color of aloe gel and commonly pasteurized aloe is marketed without adsorption on activated charcoal. This material is called 'non-decolorized' since it has not been treated (decolorized) with activated carbon. However, because it is difficult to exclude all mesophyll, most non-decolorized aloe gel undergoes some color change after batch pasteurization (see next section). The most commonly employed preservatives to prevent bacterial growth are benzoate, up to 0.1%, and sulfite, up to 0.1% for cosmetics. Sorbate in concentrations up to 0.1% is used to retard the growth of fungi. Anti-oxidants are also added in an attempt to prevent color change. Chief among these are ascorbate, up to 0.1%. It should be noted that sulfite also has anti-oxidant properties. Citrate, up to 0.2%, or other food approved acids, are usually added as a buffer to keep pH in the proper range of less than 4.5, which in itself has a mild bacteriostatic effect. Citrate itself has a slight anti-oxidant effect. In '1:1 Gel' destined for cosmetic use, Germaben II is an excellent anti-microbial preservative. Methyl paraben, propyl paraben, Germall 115 (imazolidinyl urea), either individually or in combination as Germaben II above, are the foundation of cosmetic anti-microbial preservatives.

'Aloe Vera Gel 1:1' was the first product sold by the aloe industry and even today may be the most widely sold feed stock1. It is employed in the production of drinks, with the addition of sweeteners and flavors, and in the manufacture of cosmetics. In the Complementary and Alternative Medicine community, it is generally believed to have the best biological activity. Since pasteurization should not theoretically change the composition of matter of a material, the chemical composition of '1:1 Aloe Vera Gel' should be identical to the IASC and ARF standards. In reality the chemistry and biological activity of the commercial materials vary enormously.

The drawbacks of this product are the high cost of shipping a material that is 99% water and the propensity of 'non-decolorized' aloe for color change.

Relationship of bacteriology to overprocessing

It is the firm conclusion of these authors that if aloe contains greater than one million CFU bacteria, it should be discarded and not processed. Table 8.5 illustrates one reason for this opinion — the aloe is likely to have lost biological activity. Furthermore, processing aloe with massive bacterial contamination is likely to contaminate processing equipment and the processing environment, thereby imperiling future batches. However, many companies will process it anyway, hoping that application of enough heat for a long enough period of time will kill all microorganisms. Consumer product manufacturers need to be able to identify such material so as to avoid incorporating it into their products.

A common procedure is to pasteurize heavily contaminated aloe material at a higher temperature and for a longer period of time than is usual. In batch pasteurization temperatures of 80 °C and times of 30—45 minutes are not uncommon. In HTST, temperatures of 95 °C are normally used. When heavy bacterial contamination is suspected, dwell times are increased to 5—19 minutes. If the aloe material is still not sterile after processing, it is run through pasteurization for a second time. This 'reworking' is very often accompanied by additional treatments with activated charcoal in an attempt to remove the results of the caramelization and anthraquinone oxidation that occur during prolonged heating.

The result of this is aloe material that upon organoleptic testing is called 'over processed.' Despite the use of activated charcoal there is often a yellow-brown color due to products of oxidation that are sufficiently hydrophilic to pass through the charcoal. There may be a tendency for this color to intensify and darken with time. This color

1 No precise data on production of the various A. barbadensis extracts are available. The IASC makes estimates but admits that these numbers are only guesses. The three largest producers of A. barbadensis in the world are privately held and therefore disclose neither profit nor production figures.

Table 8.9 Symptoms and Causes of 'Overprocessed' Aloe.

System

Property/Analyte Cause

Organoleptic Characteristics

Color

Oxidation of tetrahydroxyanthraquinone to 'red compound' with Subsequent condensation to brown/black polyphenolic material

Caramelization of glucose and polysaccharide by heat

Loss of aroma due to evaporation of terpenes, etc.

Loss of sweet overtones — consumption of glucose by bacteria

Production of lactic acid by bacteria

'Scorched' taste — oxidation of sugars

Absent

Color

Aroma

Taste

Taste

Laboratory Malic Acid

Findings Glucose Absent

Polysaccharide Lactic Acid

Acetylated Glucomannan absent Present change problem is particularly common in batch processes rather than HTST. Many of the terpenes and esters that give aloe its distinctive 'woody' aroma have been lost due to evaporation. These are replaced by the odors of oxidized materials reminiscent of burnt sugar. Finally, the taste of the aloe is altered. Although native aloe is somewhat bitter, it also has a sweet overtone, because approximately 10—20% of the solids are glucose. This distinctive taste is lost and the material is very bitter, with the sweetness replaced by with a pronounced 'scorched' taste.

Chemically, overprocessing is associated with changes that we discuss throughout this chapter and in frequent reviews (Pelley etal, 1998; Pelley and Strickland, 2001). These include: (i) a loss of polysaccharide content; (ii) the disappearance of glucose; (iii) the appearance of lactic acid; and, (iv) diminished to absent levels of the organic acid, malate. The normal polysaccharide content of aloe gel is about 6 to 12%, with alcohol precipitable hexose as a percentage of the total solids. 'Over-processing', or mis-processing, generally lowers polysaccharide to 1 to 2% of the total solids due to the uncontrolled activity of endogenous and exogenous P 1 ^ 4 glucosidases. Specifically, the acetylated glucomannan is hydrolysed and the only polysaccharides that remain are the galactan and pectins (Pelley etal., 1998). This loss of polysaccharide is also observed when Whole Leaf Aloe is exhaustively treated with cellulase during pre-processing to boost the yield of juice. The micrococci that cause most of the bacterial contamination of aloe appear to be very efficient at assimilating malic acid. This results in the loss of the 'E Peak' in the HPLC analysis of aloe.

It is our experience that up to half of the authentic commercial aloe in the marketplace displays some of these chemical changes and at least 20% of all products display the complete spectrum of degredative change (Pelley etal., 1998). This does not mean that these materials are not legally, 'authentic' aloe. They have not been adulterated or diluted. However, it means that these 'over processed' materials are not good quality aloe. These materials are simply aloe that has been allowed to rot during processing. This aloe has been 'over-processed', either through a lack of knowledge of proper technique, a failure to adhere to good manufacturing practices, or in order to cover up material that at one time experienced severe bacterial contamination. Proper chemical analysis, which should be done by the feedstock vendor, will inform the knowledgeable consumer product manufacturer of the actual history of the preparation.

Conclusive caveats or how a feedstock user can avoid overprocessed aloe

At the beginning of this chapter we described the production of good quality aloe gel as a conflict between Senior Management, Production and Quality Control. Advantages to Management are sometimes counterbalanced by arguments from Sales and Marketing who will tell the Consumer Product Manufacturer that:

• 'we don't have a quality problem, we haven't had to reject a batch of product in years'

• 'our leaf supplier is so good we don't have to test for bacteria in raw gel'

• 'a bacterial count before pasteurization is not necessary'

• 'sanitation is no problem. Our employees wear hairnets and gloves'

• 'if you have a problem with the product, don't dispose of it, just send it back'

• 'if the final product is clean, the type of original contamination is irrelevant'

• 'glucose and malic acid level tests are not economic'

Ideally, the vendor of an aloe feedstock should be able and willing, as part of the specification sheet, to supply the customer with:

1 Processing History. When were the leaves for this batch harvested, how long was transport, how long was preliminary and intermediate processing? Was the product ever reworked?

2 Bacterial History. Was microbiology done on the raw gel as well as on the finished product. If bacteria were found, what type of bacteria were they?

3 Chemical Testing. What is the glucose, malic acid and lactic acid content of this batch?

No aloe supplier is going to give you this willingly. Disposing of batches and laboratory tests cost money. Above all, there is a profound suspicion of science in the aloe industry. The head of one of the two largest aloe companies in the world proudly boasts that he has never spent a dollar on research and development. If we were purchasers of aloe feed stocks we would strongly consider the way we felt about the products we manufacture. If we were proud of our consumer product we would insist upon quality feed stocks and would not put overworked material into our product.

Control of aloe-associated organisms in the processing plant environment

Of all the questions that we are asked, one of the most frequent concerns processing plant sanitation. Since every plant is different we can only make general recommendations. We do, however, have preferences. First of all, we like commercial sanitizing agents that contain quaternary amino compounds (QUATS) with added surfactant. Weekly, this cleaning routine should be interrupted by the use of a different agent to avoid selecting for QUAT-resistant organisms. Our favorite agent for this is 200 ppm sodium hypochlorite solution containing 0.1% detergent. Iodophors are also excellent, though expensive.

Some general principles should be observed. Micro-organisms lodge in obscure places which should therefore be eliminated. Floors should be treated with epoxy resins and walls should be smooth and easily scrubbed down. Stainless steel can be etched by bases which provides a niche for microorganisms. A tidy work area is easier to clean so that heavily contaminated aloe does not lodge; obvious but not always observed. Cleaning in Place (CIP) is effective provided that the aloe material is kept moving. Equipment that is designed for food/dairy/brewing is not designed for handling materials with high bacterial counts (say c.108CFU/ml). Bacteria on contaminated aloes will enter every crack and blind loop not sanitized by CIP. When that happens production time is lost during cleaning. No method of sterilizing aloe will work if downstream equipment is being contaminated with an inoculum of 108 organisms/ml.

Intermediate processing — anthraquinones and spoilage

Processing aloe produces color change. At the leaf washing facility, yellow sap exudes from the cut base of the leaf and is washed away. As the sun comes out and the day heats up the yellow exudate turns to red/purple. After pasteurization at a temperature of 65 °C, the vigorously stirred aloe gel turns from light yellow to a 'pretty pink' color in 15 minutes. WLE 'guacamole' fed into the depulper turns to a brown-black. Next to microbiological spoilage, color change is the most perplexing problem with which the production staff is challenged . A decade and a half ago it was thought in the U.S. aloe industry that color change was caused by 'aloin' and that it could be solved by 'decolorizing' using activated charcoal. The reality, of course, is much more complex. Although 'decolorization' works most of the time, its scientific basis for aloe is not completely understood.

The scientific basis of color change in aloe

The U.S. aloe industry has made little or no progress in understanding the chemistry of color change in A. barbadensis gel and WLE. The U.S.-dominated aloe vera industry focuses on cosmetic and 'dietary supplement' uses of gel fillets of A. barbadensis' which are grown in the sub-tropics of the Americas. European chemists have focussed on the purgative uses of the exudate of plants grown in the African and East Indian tropics. The purgative principle is the C-glucoside of aloe emodin anthrone, referred to as bar-baloin or in its cruder form as aloin.

Reynolds (1994) established a pair of analytical systems, 2-dimensional Thin-Layer Chromatography (TLC) and C18 water/methanol gradient HPLC and analyzed freshly prepared exudates from five Aloe species and 14 museum reference samples. We have in the past used simpler TLC and water/methanol systems of lower resolving power. However, we are able to compare the two systems because the positions of aloesin and barbaloin are known in both systems. In the future, we believe that a higher resolution system, such as that of Reynolds, will be preferable to the simpler systems.

There are four elements in common between the production of the purgative exudate and the color change that occurs in commercial gel and WLE. These are: (i) the vascular elements of the mesophyll; (ii) oxygen; (iii) light; and, (iv) heat. These four factors are present in the environment in which the exudate appears. After incision, yellow juice exudes. Gradually this turns first red, then brown, then black. If the incision is made in the dark, the exudate forms, but remains yellow for several hours. Upon moving the plant into intense sunlight, the gel darkens within a short time. The involvement of the four factors in color change of the gel is evident from observations made during industrial processing, as described above. Also, over a decade ago, a crude test was developed for color change potential. This involved taking 2—3 ml of the gel being processed into a 16X 150 mm tube, making the solution basic by the addition of 0.1 ml of 1M sodium hydroxide and placing the tube for a minute in a boiling water bath, with agitation to aerate the solution. A positive test, the appearance of a pink color, indicated that all of the material with the potential to develop color change, had not yet been removed.

The next advance occurred when we developed a TLC assay for the color change compound. This happened while performing the anthraquinone periodate test (Bohme and Kreutzig, 1963) on the acetone extracts of the low molecular weight dialysates of Process A aloe gel. The sample was placed on a TLC plate which was developed, dried and then exposed to ammonia fumes at a temperature of 100 °C. One zone on the TLC plate changed color to the pink shade that developed in the color-change potential test above. Extraction of the pink spot yielded the same visible light adsorption spectrum as observed in the tube test. Using the TLC/ammonia/heat assay, it was possible to partially purify the color change compound using low pressure normal phase silica gel chromatography (Figure 8.7). The color-change-potential material (Figure 8.7, lane 4) is intermediate in its polarity between barbaloin and aloesin. The material isolated was not pure, this chromatographic region being

Figure 8.7 Anthraquinones and chromones from A. barbadensis gel and representative purified and partially purified compounds. Compounds were placed on Merck silica gel G-60 plates and developed in toluene-ethyl acetate - methanol - water - formic acid = 10:40:12:6:3. Plates were photographed under short (280 nm) or long (350 nm) wavelength UV light without any specific staining. Identity of the standards, aloesin (2 ^.g), aloin A or barbaloin (10 ^g) and aloe emodin (0.2 ^.g) were confirmed by mass spectroscopy. Other zones contained 100 ^g of extract. Extracts were replicate acetone extracts of <5,000 molecular weight cut-off membrane (MWCO) dialysates of ARF'92B Standard Sample Gel (see Colour Plate 6).

Figure 8.7 Anthraquinones and chromones from A. barbadensis gel and representative purified and partially purified compounds. Compounds were placed on Merck silica gel G-60 plates and developed in toluene-ethyl acetate - methanol - water - formic acid = 10:40:12:6:3. Plates were photographed under short (280 nm) or long (350 nm) wavelength UV light without any specific staining. Identity of the standards, aloesin (2 ^.g), aloin A or barbaloin (10 ^g) and aloe emodin (0.2 ^.g) were confirmed by mass spectroscopy. Other zones contained 100 ^g of extract. Extracts were replicate acetone extracts of <5,000 molecular weight cut-off membrane (MWCO) dialysates of ARF'92B Standard Sample Gel (see Colour Plate 6).

complex (Reynolds, 1994) The compounds isolated are consistent, upon mass fragmentation, with compounds previously described as 7-hydroxyaloin, 5-hydroxyaloin, 4-hydroxyaloin and their methyl derivatives (Rauwald and Voetig, 1982; Rauwald and Beil, 1993; Graf and Alexa, 1980). At present, we cannot determine which of these tetrahydroxyanthraquinones (THA) is responsible for color change or, indeed, whether any of them actually are the molecules involved in this change. A critical observation in favor of the involvement of 7-, 5- and or 4-hydroxyaloins in color change is the observation that when this change occurs and the products of color change convert into water-insoluble molecules, the content of 7-, 5- and/or 4-hydroxyaloins decrease.

The final understanding of the identity and mechanism of color change of aloe anthraquinones will require further research.

Decolorization with activated charcoal

Figure 8.8 below summarizes the various processes and equipment necessary to remove from aloe the anthraquinones, which contribute to color change. Most activated charcoal adsorption is done in a batch process fashion. Pasteurized aloe gel (Figure 8.8, raw material, M) is run into a mixing tank of 500 to 5,000gallon capacity (2,000 to 20,000 liters), while activated charcoal (0.05 to 2% w/v) is added with mixing (Process N). After 15 to 60 minutes the activated charcoal is removed by filtration (Figure 8.8, Process O). Theoretically, the temperature at which adsorption occurs is critical. However, in most aloe industry applications, the temperature of adsorption is determined by convenience. If adsorption is conducted as part of batch pasteurization, then activated charcoal is added immediately after the pasteurization holding period is finished and temperature reduction begins. Filtration is performed when the desired final temperature is attained. Thus adsorption may begin at a temperature of 65 °C and continue for an hour until a temperature of 45 °C is reached. At this point the product is filtered and finished. On the other hand, pasteurized gel that has been stored at 4 °C may be charcoal-treated for an hour at that temperature and then filtered. Obviously the adsorption isotherms for these two processes are very different and the first process might require ten times as much activated carbon as the second process. On the other hand, the first batch may have a much higher color change potential than the second. This is

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Aloe and Your Health

Aloe and Your Health

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