Process for water-based extraction of substances from pistachio husk

Abstract

The invention is directed to a process for the extraction of pistachio husks, the process comprising the step of extracting dried and crushed pistachio husks with an aqueous HCl solution having a pH comprised between 3.5 and 6 at a temperature comprised between 40ºC and 80ºC to obtain an extract containing secondary metabolites having antioxidant activity comprising polyphenols and flavonoids. The invention is also directed to the extract obtainable by any of the preceding claims having a polyphenols content of at least 38.0 mg GA / g of matrix, a flavonoid content of at least 3.6 mg catechin equivalent / g of matrix, and a total antioxidant power of at least 3.6% inhibition as µg of gallic acid. The extract can be used for the production of a cosmetic ingredient, a food supplement, a beverage or a pharmaceutical compound.

Description

Process for water-based extraction of substances from pistachio huskField of invention

[0001] . The present invention relates to a process for the extraction of secondary metabolites having antioxidant activity, such as polyphenols and flavonoids, for the production of an active ingredient derived from pistachio husk that can be used in particular in cosmetics, nutricosmetics, food supplements, food and drinks.State of the art

[0002] . The husk of pistachio (Pistacia ver a L.), which is the outermost layer of the fruit, namely the epicarp, is currently classified as organic waste, difficult to use because the high water content, which is more than 70%, predisposes it to rapid fermentation and contamination. Currently, during the industrial processing of pistachios, the husk, which constitutes between 35% and 45% of the fruit, is removed as one of the main wastes. This contributes to environmental pollution if disposed in uncontrolled conditions.

[0003] . The literature reports a traditional use of husk in some food formulations such as jam, as a preservative for meat, and to make mayonnaise. Although its remarkable properties and potential, and its abundant content of bioactive compounds are well known in international literature, the husk has not yet been used industrially. The use of an appropriate method for the extraction of husk’s active compounds is a key choice for the recovery of the final ingredients. Extraction procedures suitable for certain types of samples and matrices are known. The preparation of the fresh matrix and the selection of the appropriate solvent also have a great influence on the extractability of functional compounds. The use of organic solvents is a technique used to obtain extracts from a plant matrix. In the literature, studies on the pistachio husk foresee types of extraction with organic solvents used alone or mixed with water. A disadvantage of using these solvents is the high cost of the solvents and their use which can be hazardous to health, as well as the high level of pollutants and the impossibility to use the final product at "food grade" level because the techniques do not allow to completely eliminate the solvent.

[0004] . Promising methods of extraction of the pistachio husk are therefore emerging today to recover polyphenols from different plant matrices, such as microwave-assisted, ultra-sound assisted, pulsed electric field assisted and enzyme assisted extractions, with the aim of developing eco-friendly technologies. Unfortunately, in general, the application of these eco- extractions technologies at pilot level is limited due to the high capital investment required,the high operating costs and the costs of configuration, training and maintenance, as well as the difficult reproducibility of these technologies at industrial level.

[0005] . Husk water extraction is an extraction technique that is preferred for the benefits it offers. In fact, phenolic compounds, as well as polar compounds, are highly soluble not only in the organic solvents seen above, but also in a polar solvent such as water, which is the best solvent for the extraction of phenolic compounds. In addition, the use of water allows the production of natural ingredients free of chemicals, which is an increasing request by nutraceuticals and cosmetics industries. Despite the beneficial end result in terms of polyphenol content, the problems encountered when using water extractions are:The experimental conditions requiring a longer duration, up to 8 hours, and therefore require high energy costs,- the use of temperatures around room temperature promotes microbial proliferation and do not allow to pasteurize the husk extract to reduce its microbial load, thus not allowing to obtain a "food grade" extract.

[0006] . Husk water-extraction is therefore not yet considered as a viable process. There is therefore a need to develop an extraction method with high yield and resulting in a better quality product and that does not produce industrial wastes.Summary of the invention

[0007] . This problem is solved, in accordance with the present invention, by means of a process for the extraction of the pistachio husk involving extraction of the crashed and dried pistachio husks using an aqueous HC1 solution at pH comprised between 3.5 and 6.0, at a temperature comprised between 40°C and 80°C.Detailed description of the invention

[0008] . The invention is directed to a process for the extraction of pistachio husks making use of dried and milled pistachio husks. In a preferred embodiment, a raw matrix of pistachio husk is mechanically separated from the fruit by means known in technique and here not described in detail. The pistachio husk is then dried using a ventilated dryer, preferably maintained at a temperature comprised between 30 and 50°C, more preferably around 40°C, with an air flow preferably comprised between 1.0 m / s and 2.0 m / s, more preferably of about 1.5 m / sec for a sufficient time to obtain a dry final matrix with a residual water content equal to or lower than lO.Owt %, preferably equal to or lowerthan 5.5 wt%. The dried husk is then mechanically milled.

[0009] . The dried and milled pistachio husk is preferably placed in an industrial infuser or extractor made of stainless steel for food use, equipped with a heating system, a filtration system, and a stirringsystem, e.g. the system is equipped with rotating blades. Once the dried and milled pistachio husk is placed in the infuser or extractor, a pre-heated solvent is added to the pistachio mash preferably in a proportion comprised in the rage of 1 g dry matrix per 20 to 40 ml of solvent. The solvent is an aqueous solution prepared by preferably adding HC1 to deionized or distilled water until reaching the desired pH comprised in the range from 3.5 to 6.0, more preferably from 4.0 to 5.5.

[0010] . The stirring system of the infusion or extraction unit is started, the pH of the mixture is measured and, if necessary, adjusted the acidity with HC1 (food grade), until the desired pH value is reached while the temperature of the mixture is brought to the desired value. Usually, this step takes from 15’ to 60’.

[0011] . When the slurry is at the desired temperature and pH, the dried and milled husk is extracted by kinetic maceration and digestion within the extractor or infuser preferably for a time comprised between 30’ and 360’, more preferably from 45’ to 240’ at a temperature comprised between 40°C to 80°C more preferably from 50°C to 70°C, most preferably of about 60°C, with a speed rotation of the stirring blades comprised between 50 and 150 rpm, more preferably from 80 to 120 rpm, most preferably about 100 rpm. When using a temperature comprised between 40 and 50°C, the preferred extraction time is comprised between 120’ and 360’, while at temperatures comprised between 50 and 70°C, the preferred extraction time is comprised between 30’ and 180’. The most preferred extraction temperature is 60°C and the extraction time is comprised between 30’ and 90’.

[0012] . The suspension of pistachio husk in acidic water is then filtered. Optionally the extraction process is repeated a second time, charging fresh solvent in the extractor. The solution obtained from the extractor may be analysed to measure the content of polyphenols, flavonoids and the antioxidant activity.

[0013] . Preferably, the solution is then concentrated using for example a well-known falling film concentrator, particularly suitable for increasing the concentration of fruit juices and other low viscosity solutions.

[0014] . The polyphenol content in the final product, after appropriate analysis, is preferably not less than 38.0 mg GAE / g (gallic add equivalents per gram of extracted matrix), preferably not less than 43.0 GAE / g; the flavonoid content is preferably not less than 3.6 mg EC / g (catechins equivalent on gram of extracted matrix), preferably not less than 4.5 EC / g; the antioxidant power is not less than 3.6 mg GAE / g, preferably not less than 3.9 GAE / g. The use of shorter extraction times ensures that the process is done at lower costs and with greater sustainability. Furthermore, the combination of higher temperatures, shorter time and lower pH, ensures that the extract is free of bacteria and consequently ready to be used in foods, beverages, supplements, cosmetic or pharmaceutical compositions.

[0015] . Preferably, the extract according to the invention has an aerobic total plate count (TPC) equal to or lower than 1000 CFU / g wherein CFU stands for Colony Forming Units. Preferably, the total count of yeast and mould (TYMC) is equal to or lower than 100 CFU / g. Furthermore, the extract is free of pathogens such as Salmonella, E. coli, Candida, Staphylococcus.

[0016] . The pistachio husk extraction process according to the above invention generates a final product of high quality that can be used as natural active ingredient, for example in a cosmetic formulation.

[0017] . In this sense, many cosmetic products made with other vegetable ingredients can use pistachio husk extract instead. For example, husk extract obtained by the method of invention can be used to produce pharmaceutical compounds, cosmetic products with antioxidant action, anti-aging, photo protective and anti-inflammatory in the form of cream, tonics, gels etc. can be made standardized powder ingredients for the formulation of food supplements and nutraceuticals with antioxidant, anti-inflammatory and cytoprotecti ve action. Ingredients for herbal preparations or ingredients for the preparation of beverages can also be made.

[0018] . Another advantage of using acidified deionized water as a solvent in the process according to the invention is that it can also use the processing waste, which remains as a residue at the end of the implementation of the invention method, without leaving chemical residues, and with potential reuse, which further reduces the environmental impact.

[0019] . In a most preferred embodiment, the process according to the invention is provided with a ventilated dryer, an industrial stainless steel infuser or extractor for food use, equipped with a heating system, a stirring system and a filtration system, a solution of acidified deionized distilled water with the addition of HC1 in a quantity such as to reach a value of pH equal to 5, defined as solvent, wherein the method comprises the following steps: a preparation ofa raw pistachio husk matrix, b. drying of the raw matrix by keeping the dryer at a temperature of 40°C with an air flow rate of 1,5 m / sec for a time necessary to reach a residual water content of 5.5% in this raw matrix, obtaining a first intermediate product called dry matrix, c. crushing of the dry matrix, d. insertion into an infuser or extractor of the dry matrix, e. addition of the pre-heated solvent, in a proportion of 1 g dry matrix per 30 ml of this solvent, f. mixing of the dry matrix for a specified initial time, resulting in an add matrix, g. kinetic maceration and digestion of the acid matrix in an extractor or infuser for a time of 60 min at the temperature of 60°C, h. extraction from the extractor or infuser of the add matrix,i. emptying of the extractor or infuser with collection of solvent.

[0020] . A set of experiments were carried out varying temperature, pH, temperature, ratio husk / solvent and time. The results are summarized in Table 1.

[0021] . The total phenolic contents of the different extracts were determined by the Folin-Ciocalteau method compared to the calibration curve of gallic acid phenol compound used as a standard. The extracts were dissolved in 70% MeOH; to 100 pL of each sample solution, 0.2 mL Folin-Ciocalteu reagent, 2 mL ofH2O, and ImL of 15% Na2CO3 were added. After 2 h incubation at room temperature, the absorbance was measured at 765 nm. The total polyphenols were estimated as gallic add equivalent (GAE) and expressed in mg GAE / g extract. The data were obtained from the average of three independent determinations.

[0022] . The flavonoid content was measured using a colorimetric assay with modifications. A standard curve of catechin was used for quantification. Briefly ethanolic extracts (25 mL) and / or catechin standard solutions were added to EDO (100 mL). At time zero, 5% NaNO2 (7.5 mL) was added; after 5 min, 10% A1C13 (7.5 mL) was added, and at 6 min, 1 M NaOH (50 mL) was added. Each reaction mixture was then immediately diluted with H2O (60 mL) and mixed. Absorbances of the mixtures upon the development of pink color were determined as 4 = 510 nm. The total flavonoids were estimated as catechin equivalent (CE) and expressed in mg CE / g extract. The data were obtained from the average of three independent determinations.

[0023] . The free radical scavenging activity (antioxidant activity) of P. vera husk extracts was determined using the 2,2-diphenyl-l-picrylhydrazyl (DPPH) test. The same amount of each extract was dissolved in 70% MeOH to obtain final concentrations in the range of 0.0625-2 mg / mL. A volume of 0.5 mL of each sample was mixed with 3 mL of daily prepared methanol DPPH solution (0.1 mM) and incubated for 20 min at room temperature in the dark Then, the optical density change at 517 nm was measured with a spectrophotometer. The scavenging activity was measured as the decrease in absorbance of the samples versus DPPH standard solution. The results were obtained from the average of three independent experiments. In the table it is reported as % of inhibition / pg of gallic add.Table 1

[0024] . Comparing exp. 1 with exp. 2, it is possible to see that working at a too acidic pH leads to a sharp loss of antioxidant activity of the extract, despite the high content of flavonoids and polyphenols. Working at pH 3.75 (exp. 8) leads to a moderate reduction of polyphenols and flavonoids, but a very high antioxidant activity. A ratio husk / solvent of 15 is too low to allow a good extraction, as demonstrated in exp. 3 and 5, while a ratio 22.5 is already effective (exp. 7) and a value 30 is even better (exp. 1, 2, 4, 6, 8). Concerning the influence of time on the quality of the extract, it is evident that, when operating at a high temperature (e.g. 60°C) an extraction time of 60’ is sufficient to reach the maximum extraction, and by continuing the extraction up to 300’, there is a reduction in all parameters (see exp. 3 vs 5). On the contrary, when operating at lower temperature (e g. 40°C), longer extraction times a required (see exp. 4 vs exp. 6).

[0025] . Thus, depending on the temperature, preferred conditions are: for a temperature of 40-50°C, the extraction time is comprised between 120’ and 360’, while for an extraction temperature comprised between 50°C and 70°C the extraction time is comprised between 30’ and 180’ .

[0026] . As outlined above, the pH influences the type of extract. When using lower pH value (3.75), the total amount of antioxidants is higher than at higher pH. However, the amount of polyphenols and flavonoids is higher at higher pH.

Claims

Claims1. A process for the extraction of pistachio husks, the process comprising the step of extracting dried and crushed pistachio husks with an aqueous HC1 solution having a pH comprised between 3.5 and 6 at a temperature comprised between 40°C and 80°C to obtain an extract containing secondary metabolites having antioxidant activity comprising polyphenols and flavonoids.

2. The process according to claim 1, wherein the dried and crushed pistachio husks are obtained by a first step of drying pistachio husks in a ventilated dryer at a temperature comprised between 30°C and 50°C until reaching a water content in the husks equal to or lower than 7.0 wt % followed by a second step of crushing the dried husks.

3. The process according to any of claims 1-2, wherein the extraction takes place in an industrial stainless steel infuser or extractor for food use, equipped with a heating system, a stirring system and a filtration system.

4. The process according to any of claims 1-3, further comprising the step of filtering the mixture of pistachio husks and aqueous solution.

5. The process according to any of claims 1-4, wherein the extraction is performed at a temperature comprised between 50°C and 70°C for a time comprised between 30’ and 90’.

6. The process according to any of claims 1-5, wherein the amount of aqueous solution added to the extractor is comprised between 20 to 40 ml per g of dried husk.

7. The process according to any of claims 1-6, wherein the extract obtained after filtration is subjected to concentration by the use of a falling film concentrator.

8. The process according to one of the previous claims, wherein the stirring system has rotating blades, and the rotation speed of the rotating blades is comprised between 50 and 150 rpm.

9. The process according to any of the preceding claims, the process comprising the following steps: a. preparation of a raw pistachio husk matrix, b. drying of the raw matrix by keeping the dryer at a temperature of 40°C with an air flow rate of 1.5 m / sec for a time necessary to reach a residual water content of 5.5% in this raw matrix, obtaining a first intermediate product called dry matrix,c. crushing of the dry matrix, d. insertion into an infuser or extractor of the dry matrix, e. addition of the pre-heated solvent, in a proportion of 1 g dry matrix per 30 ml of this solvent, f. mixing of the dry matrix for a specified initial time, resulting in an acid matrix, g. kinetic maceration and digestion of the acid matrix in an extractor or infuser for a time of 60 min at the temperature of 60°C, h extraction from the extractor or infuser of the acid matrix, i. emptying of the extractor or infuser with collection of solvent.

10. The extract obtainable by any of the preceding claims having a polyphenols content of at least 38.0 mg GA / g of matrix, a flavonoid content of at least 3.6 mg catechin equivalent / g of matrix, and a total antioxidant power of at least 3.6% inhibition as pg of gallic acid.

11. The extract according to claim 10, having a polyphenols content of at least 43.0 mg GA / g of matrix, a flavonoid content of at least 4.5 mg catechin, equivalent / g of matrix, and a total antioxidant power of at least 3.9% inhibition as pg of gallic acid and wherein the TPC is equal to or lower than 1000 CFU / g, TYMC is equal to or lower than 100 CFU / g, and wherein the extract is free of pathogens selected from Salmonella, E. coli, Candida, Staphylococcus.

12. Use of the extract of any of claims 10 or 11 for the production of a cosmetic ingredient.

13. Use of the extract of any of claims 10 or 11 for the production of a food supplement.

14. Use of the extract of any of claims 10 or 11 for the production of a beverage.

15. Use of the extract of any of claims 10 or 11 for the production of a pharmaceutical compound.

Images