OA17321A - Method for the concentration and purification of the extract obtained from cashew pseudofruit waste and product with a high carotenoid Content. - Google Patents

Abstract

The present invention discloses a method for the concentration and purification of carotenoids from waste fibers from whole cashew juice production, under controlled conditions and without the use of any organic solvent. The process involves pre-macerating the cashew fibers with the use of cell-structure-disaggregating enzymes, which act on the fibrous tissue in combination with controlled pressing operations in successive aqueous-extraction cycles. The concentration of the crude extract obtained by maceration/pressing takes place at room temperature with the use of tangential-flow microfiltration membranes. The concentrated product is then processed using diafiltration techniques in order to purify it, thereby removing the majority of the undesirable components and promoting the microbial and biochemical degradation thereof. The final concentrate has a potential use as a coloring in foods for human consumption and in animal feed, being applicable to the areas of ready-to-drink juices and beverages owing to its considerable solubility in water.

Description

Spécification of Patent of Invention: CONCENTRATION AND PURIFICATION OF EXTRACT OBTAINED FROM CASHEW PSEUDOFRUIT WASTES AND PRODUCTS WITH A HIGH CAROTENOID CONTENT.

FIELD OF THE INVENTION

The présent invention belongs to the field of industrial processes for the extraction and purification of molécules of commercial interest, describing the production of extracts containing carotenoids and other substances of interest from wastes of cashew processing by means of techniques that involve pressing, optionally enzymatic macération and filtration (micro- or ultrafiltration), as well as diafiltration. The process proposed by the developed technology allows production of a yellow coloring extract, a product that can be obtained from industrial wastes of large-scale cashew juice production. In this way, such concentrated and purified extract of cashew carotenoids represents a value aggregation to a byproduct needed by the entire food sector to meet the worid demands of naturai colorings. Generally, yellow coloring used in Brazi! hâve a synthetic origin and available naturel yellows présent an orange coloration, differently from what is obtained by means of the présent technology, which is bright light yellow, owing to its composition containing xanthophylles, whose prédominant color is light yellow.

BACKGROUND OFTHE INVENTION

Industrial processes for the extraction and purification of molécules of commercial interest, as a coloring, an antioxidant or a molécule with spécifie properties for the use in human nutrition and diet hâve been the target of numerous sdentific and academie works as well as of requests of applications for patents worldwide.

Patent document Pl 0002359-0 discloses a process of high vacuum évaporation and distillation for extraction and concentration of liposoluble vitamins and provitamins, including carotenoids, starting from byproduct materiais deriving from plant and animal product industrialization. This state of the art document describes a process to concentrate nonsaponifiable substances, comprising of liposoluble vitamins and provitamins, growing factors and plant and animal hormones, called products of value, obtained from plant or animal products, or from wastes of the industrialization of such products, without the need for solvents. The process comprises extraction and concentration of said products by evaporation/distillation and the production of fatty adds and other high quality organic adds, by hydrolysis of the wastes obtained by distillation/evaporation.

Paten document Pl 0507797-4 describes a process and equipment for diafiltration of thickened fruit juices. The product to be filtrated is submitted to a filtration process. A first flow of wash fluid (water) and a second flow of a product permeate itself (flow driven back through the filtering means used) are added, thus the product flow is diluted by the first and second flow before entering the membranes system.

Patent document WO 2004094350 also describes a method for carotenoids extraction from plant material by means of supercritical fluid extraction. In this document, supercritical fluid extraction is processed in two steps. The first extract includes β-carotene production; the second extract may hâve a controlled β-carotene concentration and furthermore it includes substantiel quantifies of lutein.

This process (WO 2004094350) exhibits significant différences from the process presented by the présent invention, which does not include any heating or cryogénie step, and is a mass of industrial fibrous waste derived from cashew stalks pressing, however, it does not undergo trituration and/or enzymatic inactivation. The process presented describes a sequence of single operations and processes which, altogether, aims at concentrating and purifying a carotenoidenriched extract under mild conditions which maximize the yield and purity of the final product. On the other hand, the application for patent under analysis refers to the production of a purified carotenoids extract from carrots and other triturated vegetables which are thermally treated for bleaching. In this case, a set of operations and proœsses is daimed, espedally those for the production of a carotenoid-enriched mass, mainly beta-carotene, by initially produdng heating for enzymatic inactivation (bleaching), followed by successive triturations in different meshes to obtain a fine granulométrie mass to be then treated with enzymatic liquéfaction and by release of carotenoids in aqueous medium. In the following phase, this mass is applied on membranes for pre-concentration and thus filtered by one or two volumes and water to eliminate hydrosoluble compounds. At the end of such diafiltration step, the resulting mass is frozen and immediately defrosted to attain the final carotenoids concentration and the final mass is treated with flash pasteurization in order to acquire microbiological and biochemical stability.

Therefore, unlike the présent invention, a trituration system of the végétal material is described in the referred document, treating the material aggressively with respect to the content of interest and it should be noted that carotenoids are highly sensible to températures above 65 *C, even at low pH as it is indicated in the description thereof. Furthermore, unlike the document itself, the présent patent application does not include heating above this température and none of the steps of the process in general, as such procedure dégradés carotenoids molécules significantly and promûtes a loss of product quality. Moreover, in the process of the présent invention there is no need for cryoconcentration since, the process proposed by the présent invention, which involves the filtration step on microporous membranes, promûtes a volume réduction up to 30 times, and thus, it is more efficient

Similarly, patent document PI 0605425-0 describes a process of extraction and purification in sériés of active substances with colonng capadty from solid matrixes by using supercritical extraction with CO₂ for colorings production deriving from bixin from annatto. The referred process uses extraction and purification steps by means of adsorption and elution columns equipped with extractor and fractionator mounted in sériés that can be used in sériés or in batches. This process uses an aqueous solution as solvent, which at the end of the process is evaporated at températures between 30 and 700°C in order not to dégradé bixin.

The recovery of mixed carotenoids from microalgae (Dunaliella saiïna) is described in patent document WO 98/28082. This document describes a process in which collected cells are broken and the alga! suspension circulâtes in high pressure by vigorous pumping. Cells can be dehydrated by means of techniques of séparation bubble adsorption, including a flotation circuit, having a thinning zone and a concentration zone. If a higher carotenoids concentration is required, the algae concentrate can be filtered in a tangential-flow microfiltration unit, free of flocculant agents without losing carotenoids in the permeate.

Patent document Pl 0006126-3 présents an extraction process of carotenoids and other antioxidants from previously pressed and incubated plant tissues. The molécules of interest are recovered by précipitations and thermal treatments, mainly heating followed by qutck cooling; in this way, an extract containing carotenoids is recovered as a precipitate.

Patent document Pl 0416795-3 présents as subject matter an integrated process for the extraction and purification of tocopherofs, carotenoids and sterols from plant oils by means of alcohol estérification processes. The spécification of the referred patent reports as one of the advantage of the use of such a process the production of an enriched and purified product, free of factors of substantial décomposition of these components of interest, such as sugars that are elimînated by dialysis.

Besides the dted documents, patent document Pl 0103885-0 présents as subject matter the production of a cashew bagasse extract rich in pigments, obtained by a process comprising humidification of cashew bagasse waste from juice extraction, pressing of the resulting mixture, séparation of the extract from solids and concentration thereof by centrifugation and filtration operations.

The présent application for protection represents a development of the technique presented in Pl 0103885-0, having the same ownership and authorship of the présent application, by pooling it with essential steps in order to obtain a product with higher quality and by employing technologies that allow a continuous optimization of its processing.

Therefore, unlike state of the art documents, the présent development intends to complément the production of a crude extract obtained from cashew wastes with new processing technologies that involve pooling the steps of pressing, enzymatic macération and filtration techniques employing membranes in spécifie sequential steps, for the production of a final concentrated and purified extract suîtable for industrial use from which various substances of commercial interest are isolated, from colorings to dietary suppléments. Furthermore, the state of the art does not introduce an équivalent solution for processing cashew wastes crude extracts and it faits to describe a synergie process, such as the one introduced by the présent invention, in which the fruit extract or waste undergoes integrated pre-treatments, such as pressing and enzymatic macération aiming at optimizing the production of compounds of interest at the end of the process associated to final filtration and diafiltration processes, which generate a product of high purity. SUMMARY OF THE INVENTION

The présent invention describes a process for concentration and purification of carotenoids extracts from cashew stalk fibers for the use as naturel yellow coloring with the use of microfiltration diafiltration membranes. The proposed process occurs under controlled conditions without using any organic solvent, involving, optionally, pre-maceration of the cashew fibers with the use of cell-structure-disaggregating enzymes (pectinases, amylases, cellulases and hemîcellulases), acting on the fibrous tissues in combination with controlled pressing operations in successive aqueous-extraction cycles. The concentration of the unprocessed extract obtained by maceration/pressing takes place at room température by using tangential-flow micro and/or ultrafiltration ceramic membranes. The concentrated product is then treated with membrane diafiltration techniques, thereby removing the majority of the undesirable components and those favoring its détérioration. The final concentrate has a potential use as a coloring in foods for human consumption and in animal feed, being applicable in the areas of ready-to-drink juiœs and beverages owing to its considérable solubility in water. Based on the properties of some carotenoids présent in the final concentrated and purified extract, it is estimated that this material has a potential applicability in the area of dietary suppléments as provitamin A and antioxidants for food, pharmaceutical and cosmetîc industries.

BRIEF DESCRIPTION OFTHE DRAWINGS

Figure 1: it shows schematically the general steps of the process in a flowehart. The alphanumeric symbols displayed therein hâve the following correspondence:

E0: waste fibers from cashew stalk processing;

A: pressing;

M1 : addition of enzymes for macération of the extract to be pressed (optional);

E1: intermediate crude aqueous extract;

B: sieving;

E2: crude aqueous extract;

C: filtration on micro- and/or ultrafiltration and diafiltration porous membranes; and

E3: final concentrated and purified extract.

Figure 2: it shows schematically the general steps of the process in a flowchart with emphasis on the optimized step of pressing. Alphanumeric symbols displayed therein possess the following correspondence:

MO: cashew fiber mass for juice production;

AO: pressing for juice production;

SO: produced whole juice;

E0: waste fibers from cashew stalk processing;

M: Enzymatic macération (optional);

M1: addition of enzymes for macération of the extract to be pressed (optional);

A: pressing step of E0 extract;

A1 ; A2; A3; A4; A5 and A6: pressing cycles of E0 extract;

F1; F2; F3; F4; F5 and F6: waste fibers of each pressing cycle;

S1 ; S2; S3; S4; S5 and S6: aqueous extract of each pressing cycle;

E1: intermediate crude aqueous extract;

B: sieving;

E2: crude aqueous extract;

C: filtration on micro- and/or ultrafiltration and diafiltration porous membranes; and

E3: final concentrated and purified extract.

Figure 3: schematic représentation of a screw press. Alphanumeric symbols displayed therein possess the following correspondence:

E0: waste fibers from cashew stalk processing:

R: waste fibers outlets of each pressing cycle;

E1 : intermediate aqueous crude extract;

V: rotation speed control; and

F: applied force control.

Figure 4: it shows a scheme of the pilot unit of membrane diafiltration operation. The aphanumeric symbols displayed therein hâve the following correspondence:

1: water,

2: retained;

4A: feeding tank;

5A; 5B; 5C and 5D: membranes system unîtes;

3A; 3B; 3C and 3D: permeate outîets; and

4B: permeate recovery tank.

Figure 5: it shows a scheme of the pilot unit of membrane diafiltration operation detailing the equipment composing it The alphanumeric symbols displayed therein hâve the following correspondence:

3A; 3B; 3C and 3D: permeate outlets;

4A: feeding tank;

5A; 5B; 5C and 5D: membranes System unités;

6A: fluid inlet of the heat exchanger,

6B: fluid outlet of the heat exchanger;

7A; 7B and 7C: manometer;

8: thermometer;

9A: purge valve;

9B: transmembrane pressure control valve;

10: heat exchanger, and

11: pump.

Figure 6: flowchart of the permeate (Lh'¹.m'²) as a function of the volume réduction factor for different transmembrane pressures (Tmp).

Figure 7: flowchart of the permeate (Lh*¹.m'²) as a function of the concentration factor for two different situations - with and without pectinase addition - during the whole concentration step (filtration micro- and/or ultrafiltration porous membrane and dialysis).

Figure 8: it shows four charts, wherein (8-I) is the évolution of volume réduction factor (VRF), (8-II) or reduced diavolume (RD), (8-III) permeate flow (PF) density and (8-IV) the soluble solids content (SSC), respectively, as a function of time in the concentration process of E2 extract for two different situations - with and without pectinase addition.

DETAILED DESCRIPTION OFTHE INVENTION

The présent patent application of invention diffère from the state of the art in that it proposes a process combining concentration and purification by diafiltration with the usual techniques for extract purification from cashew fiber crude extracts.

The présent process allows the production on an industrial scale of a viscous and bright yellow liquid for different uses, such as natural coloring and antioxidant in food, cosmetic and pharmaceutic industry.

The subject matter of this patent application consists of a set of operations that aims at obtaining a concentrate of carotènes and xanthophyll, with emphasis on auroxanthin in cis and trans form, β-cryptoxanthin, mutatoxanthin and zeaxanthin. Following the aforementioned, βcarotene and lutein corne next in order of importance, as they hâve a composition enriched with several other carotenoids.

The proposed process comprises the following steps, corresponding to the information contained in Figure 1:

A) Obtaining a carotenoid crude aqueous extract, E1, from waste fibers from cashew stalk processing, E0, by means of pressing. In this step, enzymatic treatment is optional and it can take place by addition of cell-structure-disaggregating enzymes (pectinases, amylases, cellulases and hemicellulases) (M1);

B) Sieving of the intermediate crude extract obtained (E1) aiming at an initial standardization to avoid issues conceming an excessive increase in viscosity in the final phases of membranes processing, thus E2 crude extract is obtained;

C) Concentration of the obtained crude extract. E2, by means of techniques of microfiltration on porous membranes, reaching values of concentration factor between 2 and 30 times; and subséquent élimination of the soluble dry extract (sugars, hydrosoluble minerais and vitamins) contained in the concentrated extract through a process of diafiltration on micro- or ultrafiltration porous membranes, thus increasing the purity of présent carotenoids and promoting the microbiological and blochemical stability. The resulting product of (C) is the final concentrated extract E3.

Considering that the pH of the crude extract (E2) is already in a range between 3.5 and 5.0, there is no need for a correction of this value in order to attain a major stability of carotenoids présent in the final concentrated and purified extract.

The first step of the process (A) for producing the crude extract is based on the optimization of the process proposed by Brazilian patent document PI 0103885-0. Therefore, the described technological development involves the use of such extract for the operation/process of concentration and purification aiming to obtain a product with high carotenoid concentrations.

Before the start of the process, cashews are harvested and taken to a réception area of the processing unit, where chestnuts are withdrawn immediately, and the stalks are separated to be selected and washed for withdrawal from dirt and unwanted components for juice processing.

Afterwards, the stalks are directly pressed in an expeller-type or screw press, typical of cashew juice and cajuina processing in processing units in the Northeast of Brazil. ln this step, about 80% of whole juice and 20% of waste fibers are produced, as described in PI 0103885-0. Thus, these fibers (E0) are humidified in the ratio from one to two parts of fibers for each part of water to be pressed.

After that, the process introduced by the présent technology begins, with step (A) referring to a sériés of pressing on E0 in order to obtain the intermediate extract (E2). In this step, commercial enzymes with pectinolytic, amylolytic and cellulolytic activity may be added or not in order to increase the yield of pressing, if needed. The addition of enzymes may be up to 0.5% of the mess of fibers, the used value usually being 0.2%.

After crude extract E2 is obtained, it is taken to step (B), where it is treated in steves with meshes between 0.1 and 1.0 mm, aiming at an initial standardization to avoid issues of excessive increase in viscosity in the final phases of membranes processing.

The processing of the crude extract by filtration on microporous membranes is then carried out in (C) aiming at initially reducing the water that was added during the enzyme-assisted extraction in the previous phase, if this has occurred. Afterwards, step (C) is processed into distinct phases: a first phase (C1) in which the volume of the feeding tank (1A) is kept constant until the concentration of the crude extract reaches VRF (volume réduction factor) between 2 and 30 or until déplétion of crude extract volume to be added, wherein in this moment it begins a phase (C2) of concentration with maximum réduction of the volume of concentrated extract within the recirculation system, herein called retained, until the lowest recirculation limit in the set of membranes, ln this situation, aération of the retained should be avoided, and a volume that does not allow aération by recirculation pump should be kept. Therefore, a cold pre-concentration is promoted, using the technology of filtration on aluminum oxide microporous membranes, or simply aluminum, wherein other materials may be possibly used. These membranes hâve the capacity of totally retaining carotenoids and other hydrophobie molécules and, due to this characteristic, they are empioyed as a means of rétention of the carotenoids contained in the extract.

Therefore, in this last step (C), following C1 and C2, in which an initial preconcentration phase takes place and the concentration factor is increased until a recirculation within the System in the lowest limit of the feeding tank of the microfiltration unit, a phase C3 is processed, with the purification of the concentrated extract obtained after C1 and C2 by the execution of the process of diafiltration on microfiltration membranes.

Thus, what is retained at the end of (C2) undergoes diafiltration (C3) with volume of pure water (diavolume) added until the desired soluble solids content (°Brix) is reached.

The entire operation is carried out under température conditions between 10 e 50'C aiming at maintaining the functional properties of carotenoids that will be obtained as a concentrated solution purified by diafiltration at the end of the process.

Due to the presence of low fermentable sugars concentration, the obtained product may be stored at réfrigération températures for later use.

The final product may also be thermally treated to provide a longer durabîlity, although the use of heat is avoided along the entire process, as it deteriorates the carotenoid content, modifying its molecular profile and consequently its provitaminic and antioxidant activity. On the other hand, even if it is thermally treated, the final product has a high colonng power for use in the food and pharmaceutical field.

The carotenoid concentrate (E3) obtained has the characteristics of being an aqueous extract, in the form of a bright yellow émulsion with the capacity of coloring juices in a yellow and yellow-orange tonality for the use in food and beverages. In this way, the product may be used as a natural yellow food coloring; a natural antioxidant in the form of capsules (nutraceutics); provitamîn A and fractioning to Isolate carotenoid groups with spécifie fonctions is still possible.

With regard to step (C), it is important to characterize the pilot unit of tangential microfiltration, represented schematically in Figures 4 and 5, in which the used solvent is water.

The microfiltration unit has a set of four membranes, preferably made of aluminum oxide (Membralox*), where other types of membrane may be used optionally, In order not to limit the embodiment of the invention to this one. Its feeding tank (4A) has 3L to 10L capacity with four modules arranged in sériés, each one containing a ceramic membrane with a filtration area from 0.0001 to 0.0900 m¹, in preferred embodiments at least 0.0055 m² and a porous diameter from 0.01 pm to 0.5 pm, ideally 0.2 pm. The pressures of the four microfiltration sections are: P1 = 2.75 bar; P2 = 2.25 bar; P3 = 1.75 bar and P4 = 1.25 bar, wherein ail of them, as well as the system pressure, can vary from 0.5 to 10.0 bar. Based on the presented data, the embodiments of the invention involve resizing on the basis of the same parameters, which use the base concept to treat the desired volumes of cashew stalk wastes.

A progressive cavity pump (Moineau) whose power atteins at least 1.0 CV, being higher or lower as a fonction of the amount of matter to be processed, is represented in item (11) of Figure 5, allows liquid pressurization and its tangential-flow in the system. Tangential speed is fixed at a value from 1 m.s'¹ to 10 m.s'¹ and what is retained is continuously circulated in the filtration system. The dead volume of the system is calculated as 1.3 L for a feeding tank with a 3 L capacity, distributed in the construction éléments (pumps, pipes, membranes and tray).

The microfiltration unit allows controlling a sériés of parameters, among which:

transmembrane pressure, general température of the system, tangential speed and, as set out, the phase involving diafiltration itself follows a model comprising three phases, équivalent to C1, C2 and C3 described before: (1) pre-concentration, at constant volume (C1); (2) concentration at variable volume (C2); and (3) diafiltration/purification at constant volume (C3).

The general processing conditions for carotenoids purification from waste fibers from cashew stalk processing involve checks of different operational parameters. The température was checked by means of a heat exchanger (10), as indicated in Figure 5, installed in the redrculation circuit of what is retained by power usage in a jacket extemal to stainless pipes with cold or hot water aiming at obtaining a température stabilization at the required range, which Is from 38 to 42°C. When working with carotenoids, this température may be further broadened to a range varying from 10 to 85°C, depending on the purpose given to the final desired product Based on metabolic pathways of carotenoid dégradation, a further interest may exist in spécifie molécules that are degrading products possessing coloring activity that is more pronounced than in its natural state and, in other cases, the needs for maintaining the integrity of the molécule. Thus, according to the target product lower or higher températures are used. The tangential speed was fixed by means of rotation controi of the engine of the used pump (11), also shown in Figure 5, wherein it can be controlled through the use of frequency Inverters for increasing or decreasing this velodty as a function of the nature of the treated material (either more viscous or less viscous). And transmembrane pressure is controlled by means of a final controi valve of inlet (9A) and outlet (9B) pressure ofthe pilotsystem, indicated in Figure 5. Depending on mechanical résistance ofthe used membrane, more or less flow speed of the retained may be used, thus increasing drag and, as a conséquence, the self-deaning process of the filtration surfaces, favoring an increase of average permeate flow. In this case, an average transmembrane pressure from 1 to 5 bar in the system, preferably 2.7 bar, is recommended, due to the characteristics of the system and of the pump driving the motive power; however, such pressure can be adjusted to other values according to the needs/capadties of the production system.

The option for diafiltration system at constant volume was established with a view to optimizing the use of water as a component of diafiltration (solvent). As this natural resource is scarce in those régions where cashew is grown and the presented system promotes a reduced use of water, there is a significant gain in the process. However, the use of the diafiltration system at a variable volume for extract and cashew fibers is not exduded.

Therefore, it is crudal to note that this technology is viable for a great range of applications and equipment suitable for use, as well as operational parameters, Thus, they are not restricted to those presented in the examples and/or descriptions of the présent invention.

EXAMPLES

The présent invention is further explored by the following examples.

Example 1 — Production of purified carotenoid extract.

A crude aqueous carotenoid extract was obtained, represented in Figure 1 as E1, from waste fibers from cashew stalks processing (E0) and by means of six consecutive pressing cycles, as indicated in Figures 1 and 2 by operation (A), which includes the addition of pectinases, as shown in Figure 2 by operation (M). Water was added to the mass of cashew fibers (MO) (1:1 mass/mass) and subjected to pressing in a INCOMAP-300 model press (300 kg.h'¹ nominal capadty) for juice production, which resulted in waste crude extract (E0) and whole juice (S0). Afterwards, water and pectinases (500 mg.kg’¹ of pectinase Pectinex SP-L - Ultrazymes®) were added to waste fibers (E0) and the material was homogenized. The température was kept at values from 50 to 55°C and the same press was used (2500 N force) to submit the material to six consecutive pressing cydes, with the reincorporation of the extrads (S1 to S5) and the mass of fibers (F1 to F6) obtained in each of the pressing (A) step (A1 to A6), as illustrated in Figure 2. Figure 3 shows a schematic représentation of the press used.

After pressing, an émulsion presenting a yellow coloration due to the presence of carotenoids was obtained. Such émulsion was filtrated on stainless steel sieves with 0.30 mm mesh in order to remove suspended partides. The extract obtained after sieving, E2, is called crude aqueous extract, and was stored under réfrigération (-20°C) in packages of low density polyethylene for storage prior to concentration process (C).

The subséquent concentration step (C) employs tangential microfiltration techniques. A four-membrane system MEMBRALOX (PALL-EXEKIA), with 0.22 m² filtration area and 0.2 pm porous diameter, made of aluminum oxide of the monotubular type was used. The system pressure was 2.75 bar, the température being set at 40°C (±2°C). The process was carried out under a concentration factor equals to 13. The concentration by means of techniques of microfiltration on microporous membranes was conducted in three phases: a first phase in which the volume of the feeding tank is kept constant until the déplétion of the volume of the crude extract, wherein in this moment a concentration phase begins with maximum réduction of the volume of the concentrated extract within the redrculation system, herein called retained’, until the lowest redrculation limit in the set of membranes; and the third phase in which diafiltration on microfiltration microporous membranes takes place with the addition of pure water volume until a soluble solids content between 20 and 30 °Brix is reached.

The operation yield deriving from 1000 kg cashew stalks is about from 30 to 40 kg of concentrated diafiltrate (E3).

Total carotenoid contents of both the intermediate crude extract (E1) and the final concentrated extract (E3) were analyzed by liquid chromatography, as shown in Table 1.

Table 1 — Carotenoid content (mg.kg'¹) in the crude extract of cashew fibers (E1) obtained in six consecutive pressing cycles and in the final concentrated extract (E3).

Carotenolds	Crude Extract (E1)	Concentrated Extract (E3)
Auroxanthin (c/s and trans)	3.08	16.10
Mutatoxanthin trans	0.62	2.50
Lutein	1.04	4.70
Mutatoxanthin cis	1.25	6.30
Zeaxanthin	1.10	5.60
Cis antheraxanthin	0.20	0.80
β-Cryptoxanthin	1.52	8.00
13-ds-|3-carotene	0.13	0.70
a-Carotene	0.09	0.60
β-carotene	0.52	2.50
Others	0.85	6.40
Total Carotenoids	10.40	54.20

Example 2 — Comparison between extraction with and without the use of enzymes.

100 kg batches of crude fibers (E0) were used and pressed with an INCOMAP 300 press in six pressing cycles. The extracts were submitted to two processes: the first (I), with 500 mg.kg’¹ commercial pectinase (Pectinex SP-L- Ultrazymes*) and, the second (II), without addition of pectinase, the other operational conditions kept as the established optimized conditions provided by the process proposed by the présent invention.

In both processes of this example (I and II) 20 L of crude extract resulting from the described pressing were used, which underwent three phases in (C): pre-concentration with a decreasing concentration of feeding tank volume and; a final dîafîltratîon step. Dlafiltration begtns when the system attains a lower circulation volume of what is retained, and starting from this point, the circulating volume is kept constant until the end of the process. The system employed has a feeding tank of 3.0 L capacity.

Therefore, pre-concentration was initially carried at constant volume of feeding tank, wherein the concentration factor attained values of about 5 and after a second concentration phase with a decrease of tank volume to the least possible volume, when the concentration factor reaches its maximum value, but without damaging the carotenoid content. After reaching the maximum concentration factor in the microfiltration system, a final phase of purification by diafiltration of what is retained is conducted at the limit condition of circulating volume, which is about from 1.3 to 1.5 L in the microfiltration pilot unit

In ail the phases of the process, the adopted operational conditions were: feeding température of crude extract of 40°C (± 2), average transmembrane pressure of 2.75 bar (± 0,3) and speed of de 6.0 m.s'¹.

In this example, the parameter addressed in step (C) is explained. The used method consisted of an initial pre-concentration phase which increased the concentration factor to a system recirculation at the lowest limit of the feeding tank of the microfiltration pilot unit, followed by the purification phase of the concentrated extract by diafiltration on microfiltration membranes, as presented above. Furthermore, samples for the analysis of the global process performance were collected every 10 minutes, wherein aliquots of permeate were collected and the volumes in précision graduated test tubes submitted to physicochemical were measured. During the diafiltration phase, in each sample of permeate an équivalent volume of distilled water was replenished to keep total soluble solids content dose to zéro at the end of the process, so as to promote an increase in the purity of carotenoîds présent in the final concentrate.

Both in (I) and (II), the global process for concentration and dialysts of the 20 liters used in each of the experiments took about 10 hours per essay. Such method led to the production of a concentrated extract that could reach concentration factors in the order of 20 to 30 times when compared with the initial volume used in the concentration phase, with good performances of permeate flow kept even when high values of volume réduction factor (VRF) were reached.

Bestdes (I) and (II) essays, intermediate works were conducted in experimental phases which involved treatments with pectinolytic, cellulolytic and amylolytic enzymes with the purpose of investigating the behavior of the process when the extract is submitted or not to a pretreatment before canrying out the concentration operation. Permeate flows were high in ail treatments; however, values were more significant in treatments with pectinase and amylase. Therefore, further works with diafiltration step carried out with the use of enzymes are recommended. Permeate flows in this step still remained at the same highest levels, since diafiltration consists of adding a solvent (in this case, water was added, although other solvents may be used) to eliminate the hydrosoluble components that may produce subséquent problems, such as fermentations and chemical and biochemical interactions with the components of interest In this step, permeate flows remained in the range of 130 Lh^.m’² for treatments with enzyme and of 80 L.h‘¹.m’² for treatments without enzymes.

Table 2 shows a global average of ail the parameters observed in the process of >2— concentration and purification by diafiltration of the aqueous extract rich in carotenoids obtained from waste fibers of cashew stalks of whole juice processing for (I) and (JJ).

Table 2 - Global average of ail the observed parameters in the process of concentration and purification by diafiltration.

Parameters of the Process and Main Results	Containing 500 mg.kg*1 of pectinase (I)	Without pectinase (II)
Feeding volume (mL)	20000.00	20000.00
Feeding tank volume (mL)	3000.00	3000.00
Volume in diafiltration (mL)	1320.00	1170.00
Volume of water already used in diafiltration (mL)	5889.20	5552.60
Average permeate flow (L.h*1.m'2)	130.80	80.40
Volume Réduction Factor- VRF	19.06	17.80
Initial Soluble Dry Extract (g.L*1)	52.00	52.00
Final Soluble dry Extract (g.100 mL’1)	2.00	5.00
Initial total dry matter (g.L*1)	64.80	64.70
Final total dry matter (g.L*1)	256.80	237.80
Initial insoluble dry matter (g.L*1)	12.80	12.50
Final insoluble dry matter (g.L*1)	244.00	223.00
Total carotenoids - initial (g.L*1) *	0.38x1 O*2	0.33x1 O*2
Total carotenoids-final (g.L*1) *	0.73x10*1	0.53x10*1

* Analyses in triplicate

Additionally, the variations of the physicochemica! characteristics observed in the final product of diafiltration (final concentrated extract) by using (I) are shown in brief în Table 3. From the results, it was possible to reach in the final extract (E3) concentrations of its constituents up to 10 times higher than those contained in the crude extract (E1). Furthermore, it can be observed 10 that the synergy between the optimized pressing processes in combination with the use of enzymes plus the use of microfiltration techniques was positive, since it allowed an incrément of up to 75% in total carotenoid concentration.

Table 3- Physicochemica! characteristics ofthe final extract obtained afterdiafiltration (E3).

Physicochemlca! parameters	Values
Total soluble solids (g.100 mL'1)	0.5 to 1.5
Total dry extract (g.L'1)	237.8 to 250.5
Insoluble dry extract (g.L*1)	223 a 260
Total carotenoids (mg.L'1)	0.053 to 0.53
PH	4.5 to 4.7
Ascorbic add (g.100 mL'1)	Below détectable limit
Sugars total (g.100 mL*1)	0.2 to 1.3
Titratable addity in % of citric add (g.100 mL*1)	1.26 to 1.78
Total polyphenols in tannic add (mg.100 mL'1)	250 to 500

Example 3 - Application of purified extract as a colorinq.

Conceming the application of the concentrated and purified extract obtained at the end of the process described in Example 1, it can be seen that an addition between 0.5 and 3.0% of concentrated extract to juices and beverages is enough to reach the typical yellow coloration. Example 4 - Comparative studies on concentration step (C).

During the final step of the filtration process (C) using microporous membranes, permeate sampling was performed In the system without the substitution of the power source until the operational limit of the pilot equipment, with the least possible volume of about 1.5 liters. Starting from this point, the diafiltration step began (C3), and thus volumes of water (diavolumes) were added, the soluble solids content (°Brix) being used as a control parameter every 10 minutes.

The conclusion of the process is indicated when the soluble solids control reaches a value ranging from 0 to 10.0 g.kg*¹, indicating that water soluble materials (sugars, minera! salts and vitamins) were eliminated by the operation of diafiltration. The samples for the analysis were collected in three steps: the feeding crude extract, the permeate and what was finally retained.

Figures 4 and 5 show the details of the operation of diafiltration - step (C), in which the solvent is water. Diafiltration, as shown in Figure 2, was conducted in three phases:

• First step: pre-concentration at a constant volume of the tank of 3.0 liters and crude extract feedback until déplétion of total volume to be purified by diafiltration.

• Second step: the interruption of crude extract feedback occurs. The tank volume drops until the maximum concentration point is reached.

• Third step: this is the diafiltration phase itself, when the volume of each filtrate (permeate) is collected, and afterwards the tank is replenished with the same volume of demineralized water to reach the least soluble solids content (close to zéro) In what is finally retained.

Permeate sampling was performed only by measuring permeate flow density. From the beginning of the diafiltration phase, an ATAGO digital refractometer was used to measure total soluble solids content (°Brix), monitoring it to keep it at a value dose to zéro. All the soluble materials présent in the permeate were elimînated andonly the insoluble portion of the extract with water, enriched with carotenoids, was left inside the redrculation system.

The dedsion to perform diafiltration only in the final concentration phase was due to the fact that water consomption in these conditions may be important for the project of a factory at an industrial scale. It can be noted that, in conditions of least rétention volume of the feeding tank, diafiltration is processed more rapidly and a lot of care should be taken for aération of the manipulated product in these conditions.

During this phase, permeate extraction was compensated by the addition of distilled water to the feeding tank every 10 minutes. The solids content is the washing indicator which is measured in the permeate at each extraction. Diafiltration is interrupted when this parameter reaches values of 0 and 10 g.kg'¹ In the permeate. During diafiltration, the retained volume remains constant and the VRF does not undergo alterations.

The concentration step itself (prior diafiltration promotion) is divided into two phases: pre-concentration at constant volume of the tank, with feedback thereof with crude extract and a second step, with a continuous decrease of the extract volume to the lowest limit of the feeding tank of the pilot unit. The system normally opérâtes diafiltration considering the intake of cashew fibers in the extract, obtaining a volume of about 1500 mL at the end of the concentration step. A volume slightly higher than the lowest limit of 1000 mL was used in the pilot unit In order to avoîd unwanted contacts with air. For each one of the steps it is important to establish the volume réduction factor, as explained below.

Pre-concentration step: in order to calculate the corresponding volume réduction factor, the ratio between the volume of the extract supplied in the circuit and the retained volume was used. During this phase, the supplied volume with crude extract (V_A) equals the permeate volume (V_P) in all the process. The retained volume (V_R) corresponds to the tank volume (V_B). Therefore, in this phase the VRF is calculated as follows:

VRF = 1 + V_P/ V_B, wherein

Vp? permeate volume extracted at every sample collection (mL).

V_B: feeding tank volume (mL).

In this case, the tank volume is fixed at 3000 ml during ail the pre-concentration phase.

Concentration step: during this phase, the tank volume varies and the permeate volume is extracted continuously every 10 minutes, although this extraction is not compensated by the addition of extract to the feeding tank. Thus, the volume of the circulating product decreases more rapidly and it concentrâtes in a non-linear fashion. At this point, the calculation of the changes in the VRF changes with respect to the pre-concentration step and it is calculated by taking into account that the circulating volume decreases of the same proportion collected in the permeate volume. This means that the circulating volume represents the initial volume minus the collected volume accumulated at each permeate sampling during this phase. The calculation formula for each permeate sampling in this step is:

VRF = 1 + [ Vpc/ (V_B- V_P) ], wherein

Vpc· accumulated volume of permeate during the whole process (mL). .

Ve.· feeding tank volume (mL).

Vp.· accumulated volume of permeate during concentration phase (mL).

Purification step (dlafiltratlon): during this phase there is no concentration and the volume of the tank remains constant untii the end of diafiltration, with a soluble solids content from 0 to 10.0 g.kg'¹, measured by refractometer in each ofthe sample ofdiavolumes during diafiltration. At the end of this step, manipulation should be completed and the samples of the final retained collected for the control analysis and modeling.

As a resuit of the different analyses conducted during the essays, the évolution of the permeate flow as a function of the volume réduction factor during the step of micro- and diafiltration is presented in the chart of Figure 6, under different transmembrane pressures.

Addîtionally, as Indicated in the prior example and presented in the chart of Figure 7, the sample submitted to enzyme pre-treatment had a 130 Lh'¹.m'² average stabilization and without pre-treatment it indicates a value lower than 80 LK’.m*², thus suggesting a good industrial performance and applicability of the proposed process, even for pectinase-free samples.

Moreover, the analysis of the chart in Figure 7 indicates that pectinase treatment has a higher concentration factor, reaching values around 19, whereas the pectinase-free sample has a slightly lower value, with a concentration factor of around 17.8. Even if we take into account that the values of the concentration factor are very close, it is important to observe that the time needed for reaching such concentration level was very different for each case. The pectinase treatment showed a good performance of permeate flow that gets stabile in the pre-concentration phase (C1), whereas the one without pectinase showed a stabilized flow only at the beginning of the concentration phase (C2) with a réduction in cîrculating volume without feedback.

In order to better illustrate this fact, the charts of Figure 8 présent the general behavior of extract concentration and diafiltration as a function of the duration of the process. In this way, Figure 8 shows four charts, wherein (8-I) is the évolution of the volume réduction factor, (8-II) is the reduced diavolume, (8-III) is the density of the permeate flow and (8-1V) is the soluble solids content, respectively, as a function of time, in the concentration process of extract E2An analysis of the behavior of the concentration factor as a function of time (Figure 8-1) shows that the curve refemng to the enzyme pre-treatment process présents a growing concentration rate owing to the higher permeate flow during ali the phases of the process, when compared with the essays without enzymatic pre-treatment. This fact can be attributed to pectinase action as a factor for reducing the viscosity and decreasing the average particle size in the product.

The behavior of total soluble solids content (Figura 8-IV) shows a small incrément at the beginning of the process for both cases. It is observed that soluble solids content in both initial samples was approximately 5.2 °Brix. The reading of 5,1 a 5,2 °Brix in both cases may be attributed to the presence of water inside the microfiltration pilot unit, which is stabilized after the first crude extract feedback. The decrease in the total content of soluble solids content showed a typical behavior of diafiltration processes, wherein the final point of the adopted process corresponds to content values dose to zéro. In both cases, the end of the process took place when this parameter was enough low to avoid subséquent fermentations and not to cause any détriment to total carotenoid content.

The reduced diavolume (RD) îs a value corresponding to a ratio between the added volume of water and the redrculation volume in the system. This value is normally used for system resizing, for scheduling of industrial units. Therefore, it shall be noted in Figure 8-II that this parameter grows linearly along time, indicating good diafiltration performance.

As the diafiltration phase was carried out after the maximum concentration of the crude extract, it is observed that the volume of water needed for reducing soluble solids content is very low if compared with the initial volume of 20 liters of crude extract. Diafiltration in this example was conducted with a cîrculating volume of 1.45 L (calculated by the sum of extracted permeates) and with the addition of 5.9 L of distilled water for pectinase treatment. The cîrculating volume calculated for diafiltration when there was no addition of pectinase in the previous pressing step was 1.2 L and 5.6 L of distilled water added to reach a soluble solids content dose to zer.

The obtained results prove that the proposed technology allows the production of volume concentration factors dose to 20 and that the synergie treatment during pressing phase has a positive effect on the glc

Claims (14)

1. Process for the concentration and purification of the extract obtained from cashew pseudofruit waste and product with a high carotenoid content comprising the steps of washing cashew processing waste; successive pressing of the materials comprised of cashew waste fibers; and filtration of the total liquid phase; characterized in that it comprises the steps of:

A) optionally, enzymatic treatment of the crude aqueous extract during pressing (A);

B) sieving of the crude aqueous extract (B); and

C) concentration and purification of the aqueous extract on micro- and/or ultrafiltration microporous membranes (C).

2. Process according to claim 1 characterized in that the pressing step (A) takes place with 5 to 10 pressing cycles, preferably 6.

3. Process according to daim 1 characterized by the optional addition of commerdai enzymes with pectinolytic, amylolytic, cellulolytic and hemicellulolytic adivities in the pressing step (A).

4. Process according to daim 3 characterized in that the addition of enzymes comprises values between 0.01 and 0.5% out ofthe fiber mass (E0), preferably 0.2%.

5. Process according to daim 1 charaderized in that sieving (B) takes place in sieves having mesh between 0.1 and 1.0 mm.

6. Process according to daim 1 charaderized In that concentration (C) takes place by means of micro- and/or ultrafiltration microporous membranes, comprising steps of:

C1) addition ofthe extrad (E2) to the feedîng tank (4A) at constant volume conditions;

C2) concentration with maximum réduction of the volume of concentrated extrad within the redrculation system until the redrculation limit of the set of membranes; and

C3) diafiltration on membranes with solvent addition until attaining soluble solids content from 0 to 10.0 g.kg'¹.

7. Process according to daim 6 charaderized in that the feeding tank is maintained at constant volume in (C1) until the crude extract concentration attains values of volume concentration fador between 2 and 30 and/or until déplétion of the volume ofthe added crude extract

8. Process according to daim 6 charaderized in that the employed membranes hâve a porous diameter from 0.01 pm to 0,5 pm, preferably 0,2 pm.

9. Process according to daim 6 characterized in that the pressures of the microfiltration sections varies from 0.5 to 10.0 bar, preferably from 1.75 to 2.75 bar.

10. Process according to daim 6 charaderized in that tangential speed is fixed in 1 to 10 m.s'¹, preferably 6.0 m.s'¹.

11. System for the concentration and purification of aqueous extrads charaderized by comprising a set of microfiltration membranes, extract pumping equipment, heater exchangerfs), feeding and collection tank(s), manometer (s), thermometer(s) and control valve(s).

12. Process according to claims 1 and 11 characterized in that the steps of concentration and purification of the extract take place at température between 10°C and 85°C,

5 preferably 38°C to 42°C.

13. Product of high carotenoid content of cashew fibers characterized in that it is obtained from the process of concentration and purification of cashew pseudofruit waste extract and în that it comprises the components auroxanthin; mutatoxanthin; lutein; zeaxanthin; antheraxanthîn; β-Cryptoxanthin; 13-cis-P-carotene; α-Carotene and β-carotene and it may be

10 optionally associated with other additives and/or carotenoids of interest.

14. Product according to daim 13 characterized for being used as a coloring and/or industrial additive.