METHOD FOR OBTAINING ANTIOXIDANTS, DIET FIBER, AND OTHER NUTRIENTS FROM PLANT BY-PRODUCTS

Abstract

The present invention relates to the technical field of foods and additives that are derived from edible plants. Particularly, the present invention relates to a method for obtaining a product rich in antioxidants, dietary fiber and other nutrients from byproducts of edible plants such as dehydrated pulps and juices derived from fruits and vegetables, obtaining thus a first liquid product (first liquid phase), a second liquid product (second liquid phase), and a first and second solid phases, all of them with multiple beneficial health properties. One of the main effects observed when an individual consumes these products, is a normalization in the levels of triglycerides, total cholesterol, and HDL, and the body weight of this individual is maintained even when under a regime of fat diet.

Claims

1. A method for obtaining nutraceutical products rich in antioxidants and dietary fiber from apples, pears, and blackberry by-products, comprising the steps of: i. crushing about 10 Kg to 20 Kg of apple by-products, about 3.5 Kg to 7.0 Kg of pear by-products, and about 1.5 Kg to 3.0 Kg of blackberry by-products, thus obtaining a first mixture of a first solid phase and a first liquid phase; ii. separating said first liquid and solid phases from said first mixture, preserving the first liquid phase for its ulterior use; iii. carrying out only one step of fermentation with a yeast on said first solid phase under anaerobic conditions to achieve about 2°Bx; iv. stopping the fermentation by adding a yeast inhibitor to the fermented first solid phase, thus obtaining a second mixture of a second solid phase and a second liquid phase; and v. separating said second solid and liquid phases from said second mixture and recovering them separately.

2. The method of claim 1, wherein the second solid phase is additionally dehydrated.

3. The method of claim 1, wherein the separation of the first and second liquid and solid phases is performed by compression.

4. The method of claim 1, wherein the yeast is Saccharomyces cerevisiae and it is inoculated at a concentration of 0.05 to 0.2% (w/w) of the initial weight of the plant by-products.

5. The method of claim 1, wherein the fermentation inhibitor is sorbic acid and it is added at a concentration of 0.05% a 0.6% (w/w) of the initial weight of the plant by-products.

6. The method of claim 1, wherein the dehydration of the solid phase is performed with a temperature below 45° C. and during 24 to 48 hours.

7. A nutraceutical product rich in antioxidants and dietary fiber obtained from apples, pears, and blackberry by-products through a process comprising the steps of: i. crushing about 10 Kg to 20 Kg of apple by-products, about 3.5 Kg to 7 Kg of pear by-products, and about 1.5 Kg to 3 Kg of blackberry by-products, thus obtaining a first mixture of a first solid phase and a first liquid phase; ii. separating said first liquid and solid phases from said first mixture, preserving the first liquid phase for its ulterior use; iii. carrying out only one step of fermentation with a yeast on said first solid phase under anaerobic conditions to achieve about 2°Bx; iv. stopping the fermentation by adding a yeast inhibitor to the fermented first solid phase, thus obtaining a second mixture of a second solid phase and a second liquid phase; and v. separating said second solid and liquid phases from said second mixture and recovering them separately.

8. The product of claim 7, wherein it comprises the second liquid phase, the second solid phase, or a mixture thereof.

9. The product of claim 7, wherein the second liquid phase comprises in 100 ml of said second liquid phase 47.9 mg of phytosterols, 24 mg of polyphenols expressed as gallic acid equivalents, and 3.9 g of total dietary fiber.

10. The product of claim 7, wherein the second solid phase comprises in 100 g of the second solid phase 19.7 mg of phytosterols, 601 mg of polyphenols expressed as gallic acid equivalents, and 74.5 g of total dietary fiber.

11. The product of claim 7, wherein it is a mixture of the second liquid and solid phases containing different proportions and/or concentrations of said second liquid and solid phases.

12. The product of claim 11, wherein the mixture of the liquid and the solid phases contains a dilution 1:10 of the second liquid phase mixed in a proportion of 1:1 with the second solid phase.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0027] FIG. 1 shows a diagram of a preferred embodiment of the method for obtaining a product rich in antioxidants and dietary fiber from plant by-products.

[0028] FIG. 2 shows a graphic with the fluorescence percentages of the C. elegans populations treated with different doses of the solid phase and/or liquid phase products.

[0029] FIG. 3 shows a graphic with the reduction percentages of fluorescence in C. elegans populations treated with different doses of the solid phase and/or liquid phase products in comparison to control values (NG).

[0030] FIG. 4 shows a graphic with the weight increase in BALB/c mice treated with different diets, in percentage of the initial weight.

[0031] FIG. 5 shows a graphic with the results of the glycaemia measurements in BALB/c mice treated with different diets, in mg/dl.

[0032] FIG. 6 shows a graphic with the results of the triglycerides measurements in BALB/c mice treated with different diets, in mg/dl.

[0033] FIG. 7 shows a graphic with the results of the cholesterol measurements in BALB/c mice treated with different diets, in mg/dl.

[0034] FIG. 8 shows a graphic with the results of the HDL cholesterol measurements in BALB/c mice treated with different diets, in mg/dl.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention relates to a method for obtaining nutraceutical products low in sugars and rich in antioxidants and dietary fiber, as well as obtaining other products of commercial interest, from edible plant by-products. The method of the present invention does not use solvents that are potentially toxic to health, nor does it require expensive equipment to obtain the desired final product, and by its use, it can make the most of by-products that are used as a substrate for microbial growth. Furthermore, this method uses only one solid fermentation step with a single microorganism, unlike what is described in the state of the art, making the present method a viable alternative for optimal use of plant waste.

[0036] Through this method, all kinds of waste derived from edible plants can be used, such as dehydrated pulps and juices derived from fruits and vegetables, obtaining a first liquid product (first liquid phase), a second liquid product (second liquid phase), and a solid first and second phases, all with multiple beneficial properties for health. One of the main effects observed when an individual consumes these products is a normalization in the levels of triglycerides, total cholesterol and HDL, and the body weight of this individual is maintained even when under a fatty diet.

[0037] The edible plant by-products that are used in the present method can be obtained from any fruit and/or vegetable that the food industry has tossed away, eliminated, or discarded because they did not meet the desired characteristics for marketing, but were not in a state of decomposition. In certain preferred embodiments, a type of fruit and/or vegetable, or a mixture thereof, is used. To obtain the products with the previously mentioned beneficial properties, it is preferable to use one or more types of fruit, for example, apples, pears, blackberries, sweet cucumber, grapes, blueberries, raspberries, strawberries, tomatoes, plums, cherries, among others and not limited to the mentioned examples. Preferably, waste of apples, pears, and blackberries as used first.

[0038] The method of the present invention comprises a series of steps that show in the diagram of FIG. 1.

[0039] The first step of the method of the present invention comprises weighing and grinding (1) the by-products of the edible plants until a first mixture of a first liquid phase and a first solid phase are obtained. The grinding can be carried out by any physical method known in the state of the art, preferably by milling, preferably using a centrifugal fruit mill.

[0040] The next step of the present invention is to separate (2) the first liquid phase from the first solid phase of said first mixture, preserving the first liquid phase for later use. Preferably, the separation of said phases is carried out by compression, but any mechanical means known in the state of the art can be used for said purpose.

[0041] To make the most of all the by-products obtained through the method of the present invention, the first liquid phase obtained can be sterilized (for example, by pasteurization) and packaged for commercialization as fruit and/or vegetable juice.

[0042] On the other hand, the first solid phase undergoes a single fermentation process with a single microorganism, which will reduce the amount of sugars present in that phase. For this, a yeast inoculum is added to said solid phase and it is allowed to ferment (3) under anaerobic conditions, at a controlled temperature and for a suitable time, until said first solid phase has approximately 2°Bx (±2). In a preferred embodiment, the inoculum of said yeast is added in a ratio of 0.05% to 0.2% (w/w) with respect to the content by weight of the initial edible plants.

[0043] The yeast used in the present method is any yeast that is recognized as GRAS (generally recognized as safe), preferably a strain belonging to the genus Saccharomyces. In a preferred embodiment, the yeast used is a strain of the species Saccharomyces cerevisiae.

[0044] In a preferred embodiment, solid state fermentation can be carried out at a temperature range of between 8 and 24° C., preferably at 15° C. At that temperature, it is estimated that the sufficient time to obtain the desired Brix degrees is approximately between 4 and 5 days, shaking the culture at least once a day.

[0045] Once the first solid phase that has been fermented reaches approximately 2°Brix, the fermentation is stopped by adding a yeast inhibitor (4) and mixed, so that a second mixture of a second solid phase and a second liquid phase are obtained. In a preferred embodiment, said inhibitor is sorbic acid, but others such as benzoic acid, propionic acid, sulfurous anhydride, sodium metabisulfite, among others, can be used. After adding sorbic acid, a concentration range of between 0.05 to 0.1% (w/w) with respect to the initial weight of the by-products of edible plants, preferably in a concentration of 0.1% w/w. The mixture is homogenized and left to stand for approximately 1 hour.

[0046] Subsequently, from this second mixture, the second liquid phase and the second solid phase are separated (5). Said separation can be carried out in any way described in the state of the art, but it is preferably carried out by compression. The second liquid phase and the second solid phase are recovered, the liquid can be further concentrated by an appropriate method such as lyophilization or vacuum microwave treatment, and the solid is subjected to a low temperature dehydration process, between 42 to 53° C., to stabilize it (6), preferably 45° C., for 24 to 50 hours, preferably 48 hours, using any appropriate means such as a conventional oven with heat sink, vacuum microwave, infrared oven, among others.

[0047] A second object of the present invention is a product rich in antioxidants and dietary fiber that is obtained by carrying out the method previously described. From this method, mainly three products are obtained: a juice (corresponding to the first liquid phase), a liquid rich in phytosterols (corresponding to the second liquid phase), and a solid product (corresponding to the second fermented solid phase). Thus, through this method, it is possible to make the most of all the material from edible plants, delivering added value to each of the phases obtained.

[0048] In a preferred embodiment of the present invention, the product obtained through the disclosed method comes from the processing of apple, pear, and blackberry by-products, or a mixture thereof. The final product obtained of commercial interest can be each one of the liquid phases and the second solid phase separately, or a mixture thereof that is obtained as a result of the method of the invention.

[0049] When a mixture of apples, pears, and blackberries is used as the starting raw material, a product corresponding to the second liquid phase is obtained, which comprises 0.479 mg/ml of phytosterols, 0.024 mg equivalent of gallic acid/ml, and 390 mg/ml of total dietary fiber. From the same fruit combination, a product corresponding to the second solid phase is obtained, comprising 0.197 mg/g of phytosterols, 0.601 mg/g of gallic acid equivalents/g, and 745 mg/g of dietary fiber.

[0050] In another particular embodiment of the invention, the liquid and solid phases obtained are mixed in different proportions and/or concentrations. Preferably, the mixture of the second liquid phase and the second solid phase contains a 1:10 dilution of the second liquid phase mixed in a 1: 1 ratio with the second solid phase.

[0051] The following examples are intended to illustrate the invention and its preferred embodiments, but should not, under any circumstances, restrict the scope of the invention, which will be defined by the claims appended herein.

EXAMPLES

Example 1. Obtaining a Product Rich in Antioxidants and Low in Sugars

[0052] 10 kg of apples, 3.5 kg of pears from the Regional Supply Center (CREA) of Talca, and 1.5 kg of blackberries from the Colbun district in Chile were collected, washed, and crushed in a Recco brand juicing machine, adding 100 mL/500 grams of fruit, until obtaining a pulp. By compressing the pulp, the first liquid phase was separated from the first solid phase, thereby obtaining 1 L of the first liquid phase (juice), and 13 kg of the first solid phase.

[0053] The juice thus obtained was packaged and subsequently autoclaved under pressure conditions of 1.05 bar at 110° C. for 10 minutes.

[0054] To inoculate the solid phase, between 16 grams of yeast dissolved in 200 ml of distilled water plus 5 grams of sugar were used.

[0055] It is left at a temperature between 8 to 24° C. for 4 to 5 days, with daily agitation at least until the amount of °Brix reaches 2 or until the °Brix measurement of the previous day is repeated. Subsequently, sorbic acid is added (although other preservatives such as benzoic acid, sulfur dioxide, sodium metabisulfite, etc.) can be added at a concentration of between 0.05% to 0.1% (w/w) with respect to the initial weight of the by-products of edible plants. Subsequently, the mixture is compressed until obtaining 20% moisture of the second solid phase. The second liquid phase is stored for later use, while the second solid phase is left in ovens between 42 to 53° C. to achieve complete drying.

[0056] The second liquid phase is then concentrated in a rotary evaporator, in order to remove the alcohols, at a temperature of 55° C. at 100 rpm. Subsequently, the liquid is concentrated by different techniques such as lyophilization or by using vacuum microwaves, until the samples run out of water.

Example 2. Characterization of the First Liquid Phase and Second Solid Phase Obtained

[0057] A characterization of the nutritional content of the liquid product (first liquid phase) and the solid product (second solid phase) obtained with the method described in Example 1 was performed. First, an organoleptic analysis of the phases was carried out, the characteristics of which are described in Table 1.

TABLE-US-00001 Organoleptic characteristics of the solid and liquid phases. Characteristic Solid sample Liquid sample Appearance Non-uniform fine granulometry powder Liquid Color Red - purple Purple color liquid Flavor Sui generi Sweet and acid

TABLE-US-00002 Results of the analysis of the liquid phase. Additionally, the content of moisture, total ashes, proteins, total fat, total integrated dietary fiber, available carbohydrates, energy, and phytosterols were analyzed in both phases. Tables 2 and 3 show the results of the analysis of both phases. Parameter .sup.(2) 100 mL Moisture (g) 96.5 Ashes (g) 0.4 Proteins (g).sup.(1) 0.8 Total fat (g) No detectable (<0.05%) Total dietary fiber (g) 3.9 Insoluble dietary fiber (g) 1.8 Total soluble dietary fiber 2.1 -Soluble dietary fiber (HMW) (g) 0.1 -Soluble dietary fiber (LMW) (g) 2.0 Available carbohydrates (g) 0.5 Energy (kcal) 10 -B-Sitosterol (mg) 47.9 -Campesterol (mg) No detectable (<0.08%) -Stigmasterol (mg) No detectable (<0.08%) .sup.(1) Nitrogen to protein conversion factor used 6.25 .sup.(2) Results obtained based on ρ=1.021 g/ml (20° C.) determined in the laboratory

TABLE-US-00003 Results of the solid phase analysis Parameter 100 g Moisture (g) 11.6 Ashes (g) 1.8 Proteins (g).sup.(1) 7.2 Total fat (g) No detectable (<0.05%) Total dietary fiber (g) 74.5 Insoluble dietary fiber (g) 54.9 Total soluble dietary fiber 19.6 -Soluble dietary fiber (HMW) (g) 12.6 -Soluble dietary fiber (LMW) (g) 7.0 Available carbohydrates (g) 4.9 Energy (kcal) 88 Phytosterols -B-Sitosterol (mg) 19.7 -Campesterol (mg) No detectable (<0.08%) -Stigmasterol (mg) No detectable (<0.08%) .sup.(1) Nitrogen to protein conversion factor used 6.25

[0058] An analysis of the total polyphenol content and the oxygen radical absorption capacity (ORAC) was also carried out in both phases. The results of this analysis are shown in Table 4.

TABLE-US-00004 Results of the analysis of total polyphenols and ORAC in the liquid and solid phases. Product (Phase) Total polyphenols (equivalent mg of gallic acid/100 g or ml of sample) ORAC (equivalent .Math.mol Trolox®/100 g or ml of sample) Liquid 24 251 Solid 601 34.116

Example 3. Microbiological Analysis of the Solid Phases Obtained

[0059] A microbiological analysis of the solid products obtained by the previously described method was carried out. The presence or absence of coliforms and fecal coliforms was determined; determination of presence or absence of fungi and yeasts using the plate count technique; determination of presence or absence of aerobic mesophilic microorganisms using the plate count technique at 35° C.; and determination of Salmonella spp.

TABLE-US-00005 Determination of coliforms and fecal coliforms in solid products. NMP/ml E. coli NMP/ml Total coliforms Fecal coliforms Sample #1 < 0.30 < 0.30 < 0.30 Sample #2 < 0.30 < 0.30 < 0.30

TABLE-US-00006 Determination of aerobes, fungi, yeasts, and Salmonella spp. in solid products. Mesophilic aerobes (cfu/g) Fungi (cfu/g) Yeasts (cfu/g) Salmonella 25 g (ausencia/presencia) Sample #1 2.2 x 10.sup.2 1.0 x 10.sup.1 1.0 x 10.sup.1 Ausencia Sample #2 1.4 x 10.sup.2 1.0 x 10.sup.1 1.0 x 10.sup.1 Ausencia

Both samples # 1 and 2 of tables 5 and 6 correspond to the solid phases.

Example 4. Evaluation of the Body Fat Reducing Capacity of the Liquid and Solid Phases

[0060] It was evaluated whether the products of the solid and liquid phases had a reducing effect on body fat in Caenorhabditis elegans. The mixture of the solid and liquid phases was analyzed at different doses, and it was also included in the analysis of the liquid phase at one dose (Table 7).

[0061] For sample preparation, the liquid phase was diluted in water (1:10), and then mixed with the solid phase (1: 1). This mixture was added to the NG (Nematode Growth Medium) agar, tempered to the corresponding doses. In the case of the liquid phase, it was added on the surface of the NG agar.

TABLE-US-00007 Samples analyzed in this study Sample Dose A. Solid Phase + Liquid Phase 0.25% B. Solid Phase + Liquid Phase 0.5% C. Solid Phase + Liquid Phase 1.0% D. Solid Phase + Liquid Phase 2.0% E. Liquid Phase 1.0%

[0062] C. elegans accumulates fat in the form of drops in the intestinal and hypodermic cells that can be visualized by means of Nile Red staining (fluorescent).

[0063] The experiments were carried out with the C. elegans wild type N2 strain. Age-synchronized worms were obtained from pregnant adults, collecting the embryos in plates containing Nile Red (0.5 .Math.g/ml). The different culture media that were used were: [0064] NG medium + E. coli OP50 (control) [0065] NG medium + E. coli OP50 + Orlistat (6 .Math.g/ml) (positive control) [0066] NG medium + E. coli OP50 + Sample

[0067] The embryos were incubated under different conditions at 20° C. for 3 days. Once the young adult phase was reached, worm samples (120 worms/condition) were taken and the fluorescence emitted by spectrofluorometry was quantified. The assays were carried out in duplicate.

[0068] FIG. 2 shows the percentages of fluorescence obtained in populations of C. elegans subjected to feeding with the samples as described in Table 7. Table 8 and FIG. 3 show the percentages of fluorescence reduction obtained in each treatment with respect to the control condition (NG). The doses of samples B, C, and D resulted in a significant fluorescence reduction compared to the control conditions (15%, 10%, and 5.5% reduction, respectively) (P <0.05 and P <0.01).

TABLE-US-00008 Percentages of fluorescence reduction obtained in each treatment with respect to the control condition (NG). Samples Fat reduction of C. elegans vs. Control NG (%) P-value Orlistat (6 .Math.g/ml) 25.61 ± 2.1 0.004 A (0.25%) 0.99 ± 5.2 NS B (0.5%) 15.10 ± 3.9 0.03 C (1.0%) 10.29 ± 0.3 0.009 D (2.0%) -2.23 ± 0.3 NS E (1.0%) 5.57 ± 1.4 0.05

Example 5. Evaluation of the Effectiveness of the Liquid and Solid Phases In Decreasing Cardiovascular Risk Factors

[0069] The effect of the liquid and solid phases on the decrease of cardiovascular risk factors in mice was analyzed. To do this, different diets were prepared for the animals with different percentages of liquid, solid, or mixture phase thereof, according to what is shown in Table 9.

TABLE-US-00009 Types of diet under analysis Sample Diet DN Normal diet DG Fat diet DG S 5% Fat diet + solid phase 5% DG S 10% Fat diet + solid phase 10% DG S/L 5% Fat diet + solid and liquid phase 5% DG S/L 10% Fat diet + solid and liquid phase 10% DG L 5% Fat diet + liquid phase 5% DG L 10% Fat diet + liquid phase 10%

[0070] For the preparation of the different diets, the protocol described by Moore-Carrasco et al. 2008, Molecular Medicine Report 1(3): 401-405, May 2008 was used. Briefly, the commercial pellet was crushed and mixed with the extracts in the different proportions previously mentioned. This mixture was enriched with animal fat, vegetable fat, vegetable oil, casein peptone, and a vitamin pellet to balance the levels of vitamins and trace elements. The resulting pellets were analyzed to obtain their proximal formulation (Table 10).

TABLE-US-00010 Proximate analysis of diets Sample Moisture (%) Fat (%) Proteins (%) Ashes (%) Fiber (%) Carbohydrates (%) DN 8.2 8.0 18.9 6.1 3.0 55.8 DG 28.5 28.3 19.2 5.6 4.0 14.4 DG S 10% 27.4 29.3 15.8 4.8 4.0 18.7 DG S/L 10% 26.2 27.8 17.8 5.5 3.0 19.7 DG L 10% 31.5 27.4 16.2 5.6 2.5 16.8 DG S 5% 27.2 26.8 17.7 5.1 2.7 20.5 DG S/L 5% 27.6 26.4 18.4 5.4 2.9 19.3 DG L 5% 27.0 28.0 18.1 5.5 3.0 18.4

[0071] Female mice of BALB/c strain between 25 to 35 g were used. Each diet (individual, mix, 5% or 10%) was tested in a group of 6 animals. Animals were kept at 22 ± 2° C., in a regular 12:12 h light-dark cycle (light from 08:00 to 20:00 h). Animals were weighed every day for the first 10 days of testing, and then weighed every other day until the end of the experimental period. In addition, food intake and drinking water were measured. Access to food and water was free and bedding was changed three times a week. Each cage had a record of changes in behavior or intake that was filled out daily by the staff in charge. The aspects evaluated daily were: social behavior, piloerection, aggressiveness, anguish, anorexia and water intake. The model used to test the effectiveness of the liquid, solid, or mixed phases is the induction of cardiovascular risk factors through a hypercaloric diet. These animals, when fed with a diet that contains 25% fat, develop central obesity, hypercholesterolemia, hyperglycemia, among other disorders. At the end of the experimental period (day 40), the animals were sedated and anesthetized with an intraperitoneal injection of the mixture of Ketamine, xylazine, and acetopromazine. Once the animal was found to be in deep sleep, the peritoneal cavity was opened, blood was drawn from the abdominal aorta, and tissues such as the liver, heart, white adipose tissue (WAT), and skeletal muscle (gastrocnemius) were removed. These tissues were weighed and stored at -80° C. During the handling and administration of the different products, the animals remained calm, without alterations in their social behavior or aggressiveness between themselves or with the handlers. None of the products and combinations tested produced visible damage, irritation, or disturbances in social behavior. Neither did they present alterations in their body temperature, eating and digestive habits, not observing signs of diarrhea, pain, distress, among other evaluated parameters. There were no spontaneous deaths or signs of fights between the animals.

[0072] The administration of the different products and their mixtures in the groups of animals produced a partial improvement in some of the biochemical and anthropometric parameters. FIG. 4 shows a graph of the weight gain of the mice under the different treatments. It was observed that the animals that received the fat diet without any supplement (DG) drastically increased weight, unlike the animals that received the diet supplemented with the 5% solid/liquid phase (DG S/L 5%), which showed the smallest increase in body weight.

[0073] Visceral obesity is one of the main risk factors in the development of cardiovascular and metabolic diseases, and it is difficult to deal with given its low metabolic capacity. It is an interesting finding that the administration of the fat diet supplemented with the 5% solid/liquid reduces the accumulation of visceral fat in the treated mice.

[0074] Regarding the biochemical parameters, the administration of the diets supplemented with the 5% solid and liquid phases, shows the clearest trend towards the normalization of the parameters, equalizing the levels of triglycerides, total cholesterol, and HDL, in addition to showing a low glycemia compared to the other administered products (FIGS. 5-8).