WO2017219101A1

WO2017219101A1 - Process for sequentially extracting bioactive compounds from passion fruit bagasse and use of said bioactive compounds

Info

Publication number: WO2017219101A1
Application number: PCT/BR2016/000155
Authority: WO
Inventors: Juliane VIGANO; Julian Martinez
Original assignee: Universidade Estadual De Campinas - Unicamp
Priority date: 2016-06-24
Filing date: 2016-12-21
Publication date: 2017-12-28
Also published as: BR102016014976B1; BR102016014976A2

Abstract

The present invention describes a process for sequentially extracting bioactive extract fractions from yellow passion fruit bagasse (Passiflora edulis sp.). More specifically, the process comprises steps which involve extraction with a supercritical fluid (SFE), followed by extraction with pressurised liquids (PLE), to produce extract fractions with concentrated contents of tocotrienols, polyunsatured fatty acids, carotenoids and phenolic compounds.

Description

SEQUENTIAL EXTRACTION OF BIOACTIVE COMPOUNDS FROM PASSION FRUIT AND USE OF SUCH BIOACTIVE COMPOUNDS

Field of the invention:

[001] The invention described herein falls within the field of natural product chemistry, more specifically with regard to obtaining bioactive, and refers to a sequential extraction process using supercritical fluid and pressurized liquid to obtain. fractions of concentrated extracts in bioactive compounds of passion fruit bagasse.

Background of the Invention:

[003] Plant extracts rich in bioactive compounds have gained a significant share of the market in recent years. However, one of the desired characteristics of the extracts is the concentration of the compound of interest.

Due to this fact, lawsuits of. Extraction where the extract is concentrated during the extraction steps are very important and highly desirable.

Thus, this invention proposes a sequential extraction process using supercritical fluid and pressurized liquid to obtain fractions of concentrated extracts in bioactive compounds of passion fruit bagasse.

[005] The processes used so far for the recovery of the oil from passion fruit seeds are cold pressing and recirculation of organic solvents.

The present invention makes use of two other processes: extraction to obtain fractions of concentrated extract in different chemical compounds, namely fluid extraction, supercritical (SFE) and pressurized liquid extraction (PLE).

[007] The proposed SFE process is divided into three stages. with different operating conditions, which is essential for the completion of the last stage, the PLE.

Hitherto used techniques recover only a fraction of the extract containing the dilute polar or nonpolar characteristic compounds.

[009] The study of the article '' Svpercritical CO ₂ extraction of passion fruit (Pas.siflora eduli.s. Sp.) Ultrasound assisted seed oil ', by Barrales et al, refers to ultrasonic assisted supercritical extraction using. CO ₂ as a solvent to obtain ONE fraction of passion fruit seed oil Unlike the present invention establishes four operating conditions for obtaining four distinct classes of chemical compounds.

[010] In "Optimization of supercritical carbon dioxide extraction of Passiflora seed oil" by ZAHEDI, Gholamreza et al, supercritical extraction is simulated in neural networks using CO ₂ as a solvent and obtaining a fraction of oil from passion fruit.

In CN1710043, CN103952233 and CN103992S76, reference is made to technologies that describe obtaining only a fraction of passion fruit oil using the supercritical extraction process.

In contrast, the technology proposed in the present invention obtains four extract fractions concentrated in tocols, fatty acids, carotenoids and isolated phenolic compounds. The extracts obtained according to the present invention have biological activity and can be used directly or incorporated as ingredients in food, nutraceutical and cosmetic products.

Advantageously, the fractionated extracts obtained are concentrated in certain compounds. State-of-the-art extraction techniques perform one-step extraction, obtaining a single extract fraction, not necessarily concentrated in the thioactive compounds of. interest -

Brief Description of the Invention:

The invention described herein relates to a sequential extraction process for obtaining four portions of bioactive chemical compounds from yellow passion fruit bagasse (Passiflora edulis sp.).

[015] More specifically, this one. This process comprises steps involving extraction with supercritical fluid (SFE) followed by extraction with pressurized liquids (PLE) to obtain extract fractions concentrated in tocotrienols, polyunsaturated fatty acids, carotenoids and phenolic compounds, in which each One of these fractions is obtained under specific operating conditions during the extraction process and the sequence of these conditions follows a specific order of execution.

Brief description of the figures:

[016] For a complete and complete view of the object of this invention, the figures to which reference is made are given as follows.

[017] Figure 1 represents a typical extraction unit enabled for SFE and PLE processes. The extraction unit consists of: Vl to V-6: block valves; V-7: micrometer valve; VS: safety valve; C: air compressor; F: air filter; FC; CO ₂ filter; BR: cooling bath; BP: CO ₂ pump; BA: heating bath; EC: extraction cell; R-1: reservoir of CO ₂ ; R-2: liquid solvent reservoir; B.: HPLC pump; 1-1, 1-2 and 1-3: pressure indicators; 1-4: indicators of. temperature; IC: temperature controller; FC-1: extract collection vial; F: flow meter; R: totality.

[018] Figure Z graphically represents the response surface for (A) total tocopherol concentration and (B) total carotenoid concentration of the extracts. of passion fruit bagasse obtained in the study prior to sequential extraction.

[019] Figure 3 represents the overall and target compound yield of passion fruit bagasse extracts obtained from the sequential SFE process, where (A) is the overall yield (B). is the fatty acid yield, (C) is the tocopherol yield and (D) is the total carotenoid yield.

Detailed Description of the Invention:

[020] The present invention is a sequential extraction process for obtaining concentrated extracts in distinct chemical compounds from yellow passion fruit bagasse (Passiflora edulis sp.) Using ÇSFE supercritical fluid extraction and liquid extraction pressurized (PLE) ..

G: Said process comprises. steps of:

(a) subject the passion fruit bagasse to the first stage of supercritical fluid (SFE) extraction to obtain the tocotr.i.enols rich fraction;

b) subject the material obtained in step (a) to the second extraction step with supercritical fluids (SFE.) to obtain the fraction rich in polyunsaturated fatty acids; c) subject the material obtained in step (b) to the third supercritical fluid extraction (SFE) step for obtaining the carotenoid-rich fraction;

d) submitting the material obtained in. step (c) extraction with pressurized liquids to obtain the fraction rich in phenolic compounds.

[022] The raw material used in the process of the invention corresponds to passion fruit bagasse, composed of seeds and mucilage, resulting from the pulp separation process.

[023] Bagasse is previously dehydrated at a temperature not higher than 60 ° C long enough for moisture. less than 0.15 kg water / kg bagasse and then milled to a particle diameter of less than 2 mm;

[024] In step (a), carbon dioxide (CO ₂ ) heated to 60 to 70 ° C and pressurized to 15 to 20 MPa comes into contact with the raw material for extending the first fraction of rich extxate. in t.ocotrienols, where the amount of CO ₂ used respects the S / F fraction (where S is the solvent mass and F is the pomace mass) of up to .150.

In step (b), carbon dioxide (CO.?.) Heated to 50 to 60 ° C and pressurized to 15 to 20 MPa comes into contact with the bagasse that has passed through. step (a) for extracting the second fraction of the polyunsaturated fatty acid rich extract, wherein the amount of CO ₂ used respects the S / F fraction of up to 150.

[026] In step (c), the continuous flow of carbon dioxide (CO ₂ ) heated to 50 to 70 ° C and pressurized to 25 to 40 MPa comes into contact with the bagasse that has gone through steps (a) and ( b) for the extraction of the third fraction of the extract rich in carotenoids, wherein the amount of CO ₂ used respect the fraction of S / F to 100. In step (d), the bagasse subjected to steps (a), (b) and (c) is extracted with pressurized liquids. To this end, a liquid mixture of ethanol and water, with an ethanol concentration between 40 and 80%. m / m, heated between 60 and 80 ° C and pressurized between 5 and: 40 MPa, comes in contact with the bagasse and extracts the fourth fraction ^: from extract, rich in phenolic compounds, in which piceatanol is the majority. of ethanol used respects the S / F fraction up to 450.

[028] The order of steps is crucial in obtaining the mentioned bioactive compounds.

[029] The first three steps are based on CO ₂ density under the conditions described. Density is associated with the solvating power of CO ₂ , so if the order changes, no concentrated extracts will be obtained from the compounds already described. Already the fourth step needs to be performed after the bagasse is degreased. Otherwise, the extract will be less concentrated in phenolic compounds, since the lipid content of the bagasse hinders the obtaining of the mesfios.

[030] The extracts. Obtained in accordance with the present invention have biological activity and may be used directly or incorporated as ingredients in foods, nutraceuticals and cosmetics.

Invanation Example:

[031] In order to reach the optimal PES conditions, an experimental design was performed to test the pressure (17, 26, 35 MPa) and temperature (40, 50 and 60 ° C) variables, totaling 9 experiments, conducted in duplicate.

[032] Each experiment was conducted as follows: one. certain amount of raw material. (4 g) was weighed and placed in the extractor outlined in Figure 1; the flow The CO ₂ was released and kept in contact with the sample for 10 minutes with the V-7 valve stopped and then the valve was opened and the extract collection process started and was stopped when the S / F ratio reached value of 160.

The bed of raw material contained bulk density of 0.55 g / cm ³ . For all assays, the collected extract was analyzed for overall yield, tocopherol content, fatty acid profile and total carotenoid content. The main results of these experiments are summarized in Figure 2 and Table 1. below.

Table 1 - Influence of parameters on overall yield

tocols and carotenoids, which are 60 ° C and 17 MPa and 60 ° C and 26 MPa, respectively.

From this result, the sequence of conditions was designed, with the tocols being recovered in the first stage, as they are the most soluble compounds in supercritical COc, and the carotenoids obtained in the third stage once. that need high pressure.

[036] In view of this fact, an intermediate step was then required in which the tocols were extracted without the extraction of carotenoids. This condition was also chosen based on Figure 2 and Table 1, 50 ° C and 17 MPa, which resulted in the lowest carotenoid concentration and overall intermediate yield.

[037] To confirm the hypothesis that the three classes of compounds could be extracted in sequential steps, the SFE was performed using the temperature and pressure conditions reported above.

[038] The end. To evaluate the sequential process, the same handling conditions of the extraction equipment were used in this step. However, the same raw material was sequentially extracted under the following conditions: 60 ° C and 17 MPa; 50 ° C and 17 MPa; and 60 ° C and 26 MPa. The first two steps were performed up to 150 S / F and the third stage with 100 kg S / F solvent / kg raw material.

The extracts were analyzed for quantification of tocols, fatty acids and carotenoids and the results are presented in Figure 3 and Tables 2, 3 and 4 below. Both show that the first stage concentrated the extract in tocols, the second stage recovered fatty acids without tocols and low carotenoids and the third stage concentrated the extract on carotenoids.

Table 2 - Global yield and tocol content of passion fruit bagasse extracts obtained p.ot SFE in sequential process.

na: sample not analyzed.

^ Different letters in the same column indicate significant difference (p <0.05),

Table 4 - Total carotenoid content of e _. maracajá bagasse extracts obtained by SFE in a sequential process

¹ Different letters in the same column indicate significant difference (p <0.05).

Finally, the raw material that passed the SFE was submitted to PLE in order to obtain phenolic compounds. An experimental planning was performed contemplating the extraction temperature (50, 60 and 70 ° C) and solvent formulation (50, 75 and 100%, w / w, ethanol in water) variables, totaling 9 experiments, conducted in duplicate.

Each experiment was conducted as follows: a certain amount of raw material (3 g) was weighed and placed in the extractor; the solvent flow was pumped and kept in contact with the sample for 10 minutes with the V-7 valve shut off; thereafter, such a valve was opened and the chute process of. extract was started and stopped when the S / F ratio reached the value of ^: 120. The solvent flow was. 3 g / min

The bed of raw material contained bulk density of 0.48 g / cm ³ . For all tests, the extract. The collected data were analyzed for overall yield, total phenolic content, piceatanol content and antioxidant capacity. In order to observe the effect of the previous stages of PES, the assay with the highest total phenolic content was. reproduced using bagasse that did not pass through SFE. The main results of this step are summarized in Tables 5 and 6 below.

Table 5 - Extraction conditions, overall yield, total phenolic content and picéatanol content of extracts obtained from defatted passion fruit bagasse

(DPFB) and non-degreased (nDPPB) via PLE.

Table 4 - Antioxidant capacity of extracts obtained from extracts obtained from defatted (DPFB) and non-defatted passion fruit bagasse (nDPFB) by PLE.

'Different letters in the same column indicate significant difference (p <0.05). Based on these results it was possible to choose the fourth step of the sequential process, the PLE condition of 70 ° C and 50.% ethanol (m / m), which showed higher yields of total phenolics, piceatanol and higher values of antioxidant capacity. The comparison between the degreased bagasse, that is, that passed through the SFE stages, with the non degreased bagasse, showed that the degreased bagasse produces higher yields of phenolic compounds and piceatanol, as well as, higher antioxidant capacity. These results emphasize the importance of the sequential process in the recovery of these compounds.

[044] The methods currently employed result in obtaining a single fraction of extract from the passion fruit seed. The new technology promotes fractional obtainment of the extract, so that each fraction is concentrated in tocols, fatty acids, carotenoids and phenolic compounds, which may find specific applications.

The results indicated that sequential processes can efficiently recover four fractions of concentrated extracts.

[046] The major tocotrienol was δ-tocotrienol and the major fatty acid was linoleic acid.

[047] The main carotenoids were β-carotene and β-cryptoxanthin and the main phenolic compound was piceatanol.

[048] The contents of total tocols, total phenolic compounds and piceatanol showed a high and positive correlation with the antioxidant capacities of the fractions.

Sequentially applied SFE and PLE yielded concentrated extracts in target compounds.

Claims

1. Sequential extraction process of bioactive compounds from passion fruit bagasse comprising the steps of:

(a) subject the passion fruit bagasse to the first extraction step with. supercritical fluids (SFE) to obtain the tocotrienol-rich fraction;

b) submit the. material obtained in step (a) to the second extraction step with supercritical fluid (SFE) to obtain the fraction rich in polyunsaturated fatty acids; c) subjecting the material obtained in step (b) to the third supercritical fluid extraction (SFE) step to obtain the carotenoid rich fraction;

d) subjecting the material obtained in step (c) to extraction with pressurized liquids to obtain the phenol rich compound fraction.

Process according to Claim 1, characterized in that the passion fruit bagasse is composed of seeds and mucilage;

Process according to Claim 1 or 2, characterized in that the passion fruit bagasse has been previously dehydrated at a temperature below 60 ° C long enough to achieve a humidity of less than 0.15 kg. water / kg pomace and then ground to a particle diameter of less than 2 mm.

Process according to Claim 1, characterized in that, in step (a), carbon dioxide (CO ₂ ) is heated to 60 to 70 ° C and pressurized to 15 to 20 MPa to contact the raw material for extracting the first fraction of the extract rich in tocotrienois, where the amount of CO ₂ used meets the S / F fraction of up to 150.

Process according to Claim 1, characterized in that, in step (ta), carbon dioxide (CO ₂ ) is heated to between 50 and 60 ° C and pressurized to between 15 and 20 Mpa to contact the bagasse that went through step (a) for the extraction of _. second fraction of the extract rich in polyunsaturated fatty acids, where the amount of CO ₂ used meets the S / F fraction of up to 150.

6 .. Process according to. claim 1, characterized in that in step (c) the continuous flow of carbon dioxide (CO ₂ ) is heated between. 50 and 70 ° C and pressurized between 25 and 40 Mpa to contact the bagasse that went through steps (a) and (b) to extract the third fraction of the carotenoid rich extract, where the amount of CO ₂ used respects the S / F ^* fraction up to. 100

Process according to Claim 1, characterized in that, in step (d), the bagasse subjected to steps (a), (b) and (c) is extracted with pressurized liquids, wherein a liquid mixture of ethanol and water, with an ethanol concentration between 40 and 80% w / w, is. heated to 60 to 80 ° C and pressurized to 5 to 40 MPa to contact the bagasse and extract the fourth extract rich in phenolic compounds, preferably piceata.nol, where the amount of ethanol used respects the fraction S / F up to 450.

Use of the bioactive compounds obtained according to the process described in claims 1 to 7, characterized in that being in direct preparation or incorporation into ^foods, nutraceuticals and cosmetics.