WO2021087629A1

WO2021087629A1 - Stable anti-oxidant pigment extract enriched with anthocyanins and hydroxycinnamic acid derivatives (hcad) obtained from dyed potato pulp

Info

Publication number: WO2021087629A1
Application number: PCT/CL2019/050110
Authority: WO
Inventors: María Antonieta RUIZ MUÑOZ; Pablo Enrique CORNEJO RIVAS
Original assignee: Universidad De La Frontera
Priority date: 2019-11-05
Filing date: 2019-11-05
Publication date: 2021-05-14

Abstract

The present invention discloses a stable anti-oxidant pigment extract enriched with anthocyanins and hydroxycinnamic acid derivatives (HCAD) obtained from dyed potato pulp. The extract comprises 20-35 mg/L of petunidin-3-caffeoyl rutinoside-5-glucoside, 140-165 mg/L of petunidin-3-coumaroyl rutinoside-5-glucoside, 5-15 mg/L of petunidin-3-ferruloyl rutinoside-5-glucoside and 5 – 15 mg/L of pentunidin derivative among the anthocyanins and 10-20 mg/L of 3-caffeoylquinic acid and 180-210 mg/L of 5-caffeoylquinic acid among the hydroxycinnamic acid derivatives (HCAD). Compositions comprising said extracts for use as a functional additive and/or dye in processed foods are also provided.

Description

STABLE EXTRACT OF ANTIOXIDANT PIGMENTS ENRICHED IN ANTOCIANINS AND HYDROXYCINAMIC ACID DERIVATIVES (HCAD) OBTAINED FROM COLORED POTATO PULP

DESCRIPTIVE MEMORY FIELD OF THE INVENTION

The invention discloses a stable extract of antioxidant pigments enriched in anthocyanins and hydroxycinnamic acid derivatives (HCAD) obtained from colored potato pulp and its use as a functional additive and / or colorant in processed foods, and the production process. BACKGROUND OF THE INVENTION

The current lifestyle has triggered an increase in metabolic diseases, which represent one of the main causes of death, resulting in a public health problem.

One of the main causes associated with these metabolic pathologies is eating habits, since the consumption of healthy foods has been replaced by processed foods with high levels of carbohydrates, saturated fats, salt and additives, affecting worrying levels of obesity, the main factor risk of metabolic syndrome, which in turn can trigger irreversible diseases.

Therefore, it is important to modify eating habits, incorporating healthy foods into the diet, which, although they could be added as functional ingredients, is a plus from the productive point of view that they also provide other properties to the food, like, for example, coloring.

Thus the present invention relates to the food industry. In particular, it is related to a stable extract of pigments enriched in anthocyanins and hydroxycinnamic acid derivatives (HCAD) obtained from colored potato pulp and its use as a functional additive and / or colorants in the food industry. STATE OF THE ART

Currently berries are the most used source to obtain anthocyanins, in most cases their distribution is restricted to the skin of the fruit. Thus, in cases where the anthocyanin concentration was in similar ranges, the volume of fruit to be processed into berries would be considerably high, since it would be extracted mainly from the skin of the fruits. Thus, for example agronomically, the maximum yield of blueberries is up to 3 kg per plant reaching about 13 tons / ha, while that of potatoes is 20 tons / ha. in areas of marginal yield, being able to reach in normal conditions up to more than 60 ton / ha. The harvest of berries requires a lot of labor, on the other hand, in the case of potatoes it is even possible to automate their harvesting, which is a huge advantage for obtaining pigments from colored potato pulp, being able to be obtained in substantially larger quantities due to their yield and concentration in pulp, adding to ostensibly lower costs in agronomic management, which represents an opportunity with a high potential to obtain this type of pigments, feasible to be used in a series of industries, mainly food. In the state of the art, there are also various documents which describe the use of sweet potato / purple sweet potato as a source of anthocyanins, such as publication WO2018066575, where they show that they are useful as a purple pigment and a purified product thereof, however, This document mentions genome editing by inserting an LTR-type transposon in at least two positions, which is far from the objective of exploiting selected varieties of colored potato pulp to obtain anthocyanins at a lower cost than berry crops or berries.

On the other hand, Inostroza-Blancheteau et al (Metabolomics. 2018 Oct 4; 14 (10): 138. Doi: 10.1007 / s11306-018-1428-7) analyzed native Chilean potatoes and observed differential levels of antioxidant activity and compounds phenolics between skin and pulp compared to a common commercial potato. A network correlation analysis revealed different metabolite associations between tuber tissues indicating distinct associations between levels of primary metabolites and anthocyanins, and antioxidant activity in skin and pulp tissues. In this document the present invention is not anticipated, since the purpose is to study the composition of native potatoes, and an application derived from it is not indicated nor the elaboration of a highly stable anthocyanin extract, as is done in the present invention. . For their part, Ruiz et al. (Food Chemistry, 2018, vol. 268, p. 577-584) analyzed the phenolic composition of colored potato pulp (Solanum tuberosum), highlighting its anthocyanin composition, detecting three derivatives of hydroxycinnamic acids (HCAD) in these pigmented potatoes, corresponding to the trans isomers of 3, 4 and 5-caffeoylquinic acids. In the analysis, they highlight that the concentrations of HCAD increased by 493% compared to those of fresh potatoes after frying, observing the same trend for total phenols and antioxidant activity, which is beneficial compared to the colorless varieties. This study is therefore analytical-empirical, observing the behavior of the components in the potato itself once cooked, without pursuing the preparation of extracts of the anthocyanins studied, much less the search for stable applications or formulations for food.

In 2016, Zapata et al. (Vitae, 2016, Vol. 23, No 3, p. 173-183) studied the stability of anthocyanins during the storage of blueberry juices and it was concluded that the pasteurization treatment caused a decrease of 28.5% in the concentration of Initial Total Monomeric Anthocyanins from Cranberry Juices. The stability of anthocyanins during storage was higher in pasteurized juices, being higher when stored at 0 ° C; while in pasteurized juices stored at -18 ° C anthocyanins showed less stability. Thus, this document only focuses on the anthocyanins naturally present in blueberries and their stability once the juice has been made and stored at different temperatures, showing their limited stability against storage at sub-zero temperatures.

The need therefore remains for an inexpensive source of anthocyanins and hydroxycinnamic acids (HCAD), or a composition based on them that at the same time has beneficial characteristics, such as color stability, especially in sub-zero temperatures, maintaining the antioxidant properties of these pigments under various storage conditions once incorporated into the final product.

SOLUTION TO THE TECHNICAL PROBLEM

To provide a stable extract of antioxidant pigments enriched in anthocyanins and hydroxycinnamic acid derivatives (HCAD) obtained from colored potato pulp that is applicable in the food industry, which can be used as a highly stable natural colorant under various light conditions, temperature and pH. The extract also has beneficial properties mainly due to its antioxidant capacity and proven effects on human health. The main technical contribution of the extract of the present invention is its high stability that allows it to maintain its antioxidant capacity at low temperature, preferably between 4 ° C to -20 ° C, at different pH, preferably between pH 1 and 5. Contrary to that reported by Zapata for berry extracts, which would lose their stability at temperatures below 0 ° C.

In addition, among the additional advantages is the cost associated with the production of anthocyanin-type pigments, which is significantly lower than obtaining it from blueberries, due to the higher yield of the potato crop, lower production costs per hectare, but also due to extraction costs, degree of purification and type of final product. Thus, the cost of the pigment obtained from colored potato pulp is a range between 8 - 16 times lower than that obtained from berries.

DESCRIPTION OF THE FIGURES

Figure 1 shows the basic statistical graphs between the scores of judges and treatments for the attributes of the sensory evaluation for the natural milk samples a) appearance, b) smell, c) color d) texture and e) taste.

Figure 2 shows the basic statistical graphs between the score of judges and treatments for the attributes of the sensory evaluation for the natural yogurt samples a) appearance, b) smell, c) color d) texture and e) taste.

DESCRIPTION OF THE INVENTION

The invention discloses a stable extract of antioxidant pigments enriched in anthocyanins and hydroxycinnamic acid derivatives (HCAD) obtained from colored potato pulp. These extracts can be incorporated into processed foods, such as ice cream, milk or yogurt as colorants at various color levels. The variety of colors that can be obtained is wide and ranges from reddish to violet tones, depending on the genotype of potato used and the storage pH, which can vary between pH 1 to 5. Potatoes with purple pulp have the same anthocyanin profile, varying its concentration according to the intensity of the pulp color.

The method of the present invention has the following stages: a) Washing, cutting and grinding the amount of potato to be processed. b) Mix the ground potato from step a) with extraction solvent in an acid medium in a ratio of ground potato to solvent 1: 2 calculated in grams and mL, respectively and protect the mixture from light. Mixing is done at room temperature, generally 25 ° C. c) Apply ultrasound to the mixture from step b) with a bar at 40% amplitude for 45 to 120 seconds, preferably for 60 seconds, at room temperature. d) Shake the mixture from step c) in an orbital shaker, between 100 to 250 rpm, for 45 to 90 minutes, preferably for 60 minutes at a temperature between 20 to 25 ° C. e) Centrifuge the mixture from step d) to separate the liquid extract from the solid phase between 2000 to 4000 rpm for 5 to 20 min, at 4 ° C. f) Collect the supernatant or liquid extract from step e) and transfer it to a graduated container and keep it protected from light.

Repeat steps c) to f) for at least 3 times with the solid phase obtained in f) to extract the maximum possible pigment and then continue.

Measure total volume to determine the concentration of pigments in the extract.

Store extract between -20 ° C to 4 ° C, in an acidic pH range between 1 to 5.

Optionally, preconcentration and / or lyophilization steps of the extract are carried out. The liquid extract is preferably made without further concentration.

Where the extraction solvent used in step b) is a mixture of absolute ethanol in an acid medium (CH ₃ COOH at 15%).

The liquid extract obtained from colored potato pulp comprises anthocyanins and other compounds that provide antioxidant activity such as some hydrocinnamic acid derivatives (HCAD), in addition to the extraction solvent. The anthocyanin content per kg of fresh potato can reach 1.21 g, present in all the pulp of the fruit and not only in the skin, as occurs in berríes.

Thus the invention provides a colored potato pulp extract for use as a functional additive and food coloring, comprising anthocyanins and hydroxycinnamic acid derivatives (HCAD) as main active compounds. Among the main anthocyanins present in the extract are: petunidin-3-caffeoylrutinoside-5-glucoside, petunidin-3-coumaroylrutinoside-5-glucoside, petunidin-3-feruloylrutinoside-5-glucoside and another derivative of petunidine.

Among the main hydrocinnamic acid derivatives (HCAD) present in the extract are: 3-caffeoilquinic acid and 5-caffeoilquinic acid.

The potato pulp extract has the following profile of concentrations of antioxidant compounds as shown in Tables 1 and 2:

Table 1: Average anthocyanin concentration range in colored potato pulp extract

Table 2: Average concentration range of Hydroxycinnamic Acids in colored potato pulp extract

These extracts show high stability in the dark between 4 ° C to -20 ° C at different pH values evaluated between pH 1 and pH 5, for 17 weeks, maintaining their antioxidant capacity.

APPLICATION EXAMPLES Below, some examples of embodiment of the invention are included that should be considered illustrative and not limiting thereof.

EXAMPLE 1 :

Obtaining extract from colored potato pulp. 300 g of CB2011-104 genotype potato were washed, chopped and ground. 600 ml_ of absolute ethanol in acid medium (CH3COOH at 15%) were added and the flask containing the mixture was wrapped with aluminum foil, taking care to keep it protected from light.

Bar ultrasound was applied at 40% amplitude for 60 seconds and subsequently the mixture was kept in an orbital shaker for 1 hour at 200 rpm at 22 ° C.

The mixture was transferred to centrifuge tubes and centrifuged at 3000 rpm for 10 minutes, collecting the supernatant, which was kept in a container protected from light.

After the first extraction stage, about 55% of the compounds were obtained, and with the second stage, about 80% of the compounds were extracted, so the extraction steps were repeated a third time to increase the yield.

An extract comprising 200mg / ml_ of anthocyanins and 205 mg / ml of hydroxycinnamic acids was obtained according to what is shown in Tables 3 and 4, respectively.

Table 3: Example of anthocyanin concentrations in a colored potato pulp extract

Table 4: Example of concentrations of Hydroxycinnamic Acids in a colored potato pulp extract

EXAMPLE 2:

Stability evaluation of the extract obtained.

After knowing the concentrations of anthocyanins and hydroxycinnamic acids of the invention obtained in Example 1, the stability and antioxidant capacity of the extract under different conditions of temperature, light and pH were studied. The results are shown in Tables 5, 6 and 7.

The results show that the extract of the present invention has a great stability at different pH (between 1 to 5) and in a wide temperature range (between 4 and -20 ° C) refrigerated or frozen, where it has been advantageously found that the extract is stable at - 20 ° C.

In the case of anthocyanin stability, Table 5, with respect to the concentration in the original extract, in all the extracts stored at -20 ° C more than 95% of the anthocyanins are maintained independent of the pH after 4 months of study. In the case of the refrigerated extract, only 77% remains. While, in the extract at room temperature, 23 and 40% of the anthocyanins are maintained depending on whether the extract is stored in the presence of light or in the dark, respectively.

Table 5: Total anthocyanin concentrations (g / kg) in liquid extract, study carried out over four months.

Table 6: Concentrations of total hydroxycinnamic acids (g / kg) in liquid extract, study carried out during four months.

Table 7: Total antioxidant activity (pmol / g) in liquid extract, study carried out over four months

For hydroxycinnamic acids, Table 6, in all conditions it is possible on 91% of these compounds (the value of 91% corresponds to storage at pH 1, which was the one that presented the lowest observed stability). The results for the antioxidant activity, Table 7, show that said activity remains practically constant throughout the evaluated storage period.

Without wanting to limit ourselves by theory, we consider that the superior stability of the extracts obtained from colored potato pulp with respect to the lower stability in time reported in berries, is due to the fact that the anthocyanins of the potato present a high degree of conjugation or glycosidation. , that is, they are in a glycosidized and adiated state, this union to phenolic acids and large sugars, added to the type of processing used for the extraction, which would not damage the conjugated molecules, would allow to keep the anthocyanins protected in the extracts of the present invention. The presence of hydroxycinnamic acids, antioxidant compounds that are co-extracted with anthocyanins, would also contribute to stability. As can be seen in the results, the anthocyanin concentration remains practically constant in all refrigeration conditions (between 4 ° C and -20 ° C), declining only at room temperature. Hydroxycinnamic acids decay only in light and room temperature conditions, not in room temperature and darkness, while the antioxidant capacity remains constant in all the evaluated conditions.

EXAMPLE 3:

Example 3 shows the comparative values in extracts from colored and uncolored potato pulp and blueberries. Uncolored potatoes and different types of colored potatoes were evaluated to evaluate the production efficiency of anthocyanin-type pigments compared to blueberry, based on a high theoretical agronomic yield.

From the analysis it is evident (see table 8) that the pigment content and the yield per hectare is an order of magnitude lower in blueberries, being evident that the costs in the latter are an order of magnitude higher per kg of pigment obtained. .

Table 8: Crop yield and pigment content.

EXAMPLE 4

Potato pulp extract for use in food

The present invention further provides a colored potato pulp extract for use as a functional additive and food coloring, which comprises the colored potato pulp extract.

The extract is presented in liquid form, where the extract is pre-concentrated 5-20 times, preferably 10 times (10X) to be added to food.

Use in the manufacture of additive for processed foods

The extract of the present invention can be added to processed foods, such as yogurts, cultured milk and other flavored dairy products.

Different proportions of the liquid extract were evaluated, by means of a sensory analysis panel, in order to determine the optimal proportion of color without disturbing the taste of the final product.

The optimal proportion for a dairy product (milk or yogurt) to achieve a color similar to that used in the blackberry flavor was 1 mL of 10X pre-concentrated extract in 25 mL of product.

The presence of solanine (alkaloid with toxic properties that can be found in potatoes) was also evaluated and was not detected in the extracts obtained according to the present invention.

Although most of the tests have been done on dairy products, the extracts of the present invention can be used in any food that is stored at acidic pH and low temperatures, as described above.

On the other hand, the extract of the present invention, due to its pigment and antioxidant properties, can be used in addition to the food industry, in other industries including the textile, cosmetic and paint manufacturing industries.

Sensory analysis of dairy products:

Natural milk samples or treatments were coded as follows:

YZ = sample of natural milk x (incorporation of pre-concentrated extract 10 times) ST = sample of natural milk and (incorporation of original extract)

The samples or treatments of natural yogurt were coded as follows:

XB = sample of natural yogurt y (incorporation of pre-concentrated extract 10 times)

LA = sample of natural yogurt x (incorporation of original extract)

Milk sensory evaluation results:

Figure 1 shows the results obtained for the scores given by the judges in the sensory evaluation of the YZ and ST treatments, samples X and Y of natural milk respectively.

The scores obtained for appearance (figure 1 a) and table 9) were slightly higher for treatment YZ (sample x) than for treatment ST (sample y). Statistical analyzes show that YZ treatment does not differ significantly from ST treatment (p = 0.295) in appearance. Both samples obtained averages 4, which qualifies them as good in this evaluated parameter.

For the odor attribute (figure 1 b) and table 9), the scores that are repeated the most are higher for the YZ sample, with a statistically significant difference between the YZ and ST samples (p = 0.002013). The highest average was 3.8 for the YZ sample, which evaluates it as close to good for odor.

The scores obtained for color (figure 1 c) and table 9) were higher for treatment YZ (sample x) than for treatment ST (sample y), statistical analyzes show that treatment YZ differs significantly from treatment ST (p = 0.049) in terms of color. The averages were 4.5 for the YZ sample and 4.0 for the ST sample, which qualifies them as good in this evaluated parameter.

The scores that are repeated the most for the texture attribute (figure 1 d) and table 9) were higher for treatment YZ (sample x) than for treatment ST (sample y), but the statistical analyzes show that treatment YZ does not differ significantly from the ST treatment (p = 0.055) in terms of texture. The averages were 4.4 for the YZ sample and 3.9 for the ST sample, which qualifies them as good in this evaluated parameter.

For the flavor attribute, the scores obtained that are repeated the most (figure 1 e) and table 1) were higher for treatment YZ (sample x) than for treatment ST (sample y) respectively. Statistical analyzes show that YZ treatment differs significantly from ST treatment (p = 1,308e-06) in terms of taste. The averages obtained were 4.2 for the YZ sample and 2.5 for the ST sample, which qualifies the YZ sample as good in taste and the ST sample as deficient in this evaluated parameter. Table 9. Averages of scores obtained for each attribute in milk samples.

Yogurt sensory evaluation results:

Figure 2 and table 10 show the results obtained for the scores given by the judges in the sensory evaluation of the samples or treatments XB and LA, samples Y and X of natural yogurt respectively. The scores that are repeated the most for the appearance attribute are very close to 4 in both XB and LA treatments (figure 2 a) and table 10). Statistical analyzes show that there is no significant difference between XB treatments (p = 0.05) in terms of appearance. The averages obtained were 4.1 for sample XB and 4.5 for sample LA, which qualifies them as good for the appearance attribute. For the odor attribute (figure 2 b) and table 10), the scores that are repeated the most are higher for sample XB (sample y), with a statistically significant difference between samples XB and LA (p = 0.01103). The highest average was 4.6 for the XB sample (sample y), which evaluates it close to the maximum score 5, which it describes as very good in this evaluated parameter. The LA sample (sample x) obtained an average score of 3.8, which qualifies it close to score 4 that describes the sample as good for odor.

For color, although there were differences in the scores that were repeated the most (figure 2 c) and table 10), being higher for treatment LA (sample x) than for treatment XB (sample y), statistical analyzes show that this The difference is not statistically significant between the treatments (p = 0.4498) in terms of color. The averages were 4.5 for the XB sample and 4.6 for the LA sample, which qualifies them close to the maximum score that is 5, which describes them as very good in this evaluated parameter. The scores that are repeated the most for the texture attribute (figure 2 d) and table 10) were higher for treatment XB (sample y) than for treatment LA (sample x), but the statistical analyzes show that this difference does not differ significantly. between treatments (p = 0.055) in terms of texture. The averages obtained were 4.5 for sample XB and 4.1 for sample LA, which qualifies them as good in this evaluated parameter.

For the flavor attribute, the scores obtained that are repeated the most for both samples or treatments are close to 4 (figure 2 c) and table 10). Statistical analyzes show that between treatments XB and LA there is no significant difference (p = 0.8617) in terms of taste. The averages obtained were equal, 3.9 for both samples, which qualifies them as good for flavor attribute.

Table 10. Averages of the scores obtained for each attribute in the natural yogurt samples evaluated.

Claims

1 . An extract of pigments and antioxidants prepared from colored potato pulp stable at low temperatures CHARACTERIZED because it is enriched in anthocyanins and hydroxycinnamic acid derivatives (HCAD).

2. The extract according to claim 1, CHARACTERIZED in that the anthocyanins comprise petunidin-3-caffeoylrutinoside-5-glucoside, petunidin-3-coumaroylrutinoside-5-glucoside, petunidin-3-feruloylrutinoside-5-glucoside and derivative of petunidin.

3. The extract according to claim 2, CHARACTERIZED because it comprises 20 - 35 mg / L of petunidin-3-caffeoylrutinoside-5-glucoside, 140 - 165 mg / L of petunidin-3-coumaroylrutinoside-5-glucoside, 5 - 15 mg / L of petunidin-3-feruloylrutinosido-5-glucoside and 5 - 15 mg / L of petunidine derivative.

4. The extract according to claim 1, CHARACTERIZED in that the hydroxycinnamic acid derivatives (HCAD) comprise 3-caffeoylquinic acid and 5-caffeoylquinic acid.

The extract according to claim 4, CHARACTERIZED in that it comprises 10-20 mg / L of 3-caffeoylkinic acid and 180-210 mg / L of 5-caffeoylquinic acid.

6. The extract according to claims 1 to 5, CHARACTERIZED in that the extract is liquid.

7. A process for making the extract according to claim 1,

CHARACTERIZED because it comprises the stages of: a) washing, cutting and grinding the amount of potato necessary to process; b) add extraction solvent in an acid medium (CH ₃ COOH at 15%) in a ratio of ground potato to solvent 1: 2 (grams and mL, respectively) and protect the mixture from light; c) apply ultrasound for 45 to 120 seconds; d) stir between 100 to 250 rpm for 45 to 90 minutes; e) centrifugation to separate the extract from the solid phase; Y f) Collect the supernatant and keep it protected from light.

8. The process according to claim 7, CHARACTERIZED in that the extraction solvent of step b) is absolute ethanol.

9. The process according to claims 7 and 8, CHARACTERIZED in that steps c) to step f) are repeated at least three times with the solid phase obtained in f).

10. The process according to claims 7 to 9, CHARACTERIZED in that it also comprises a subsequent concentration stage, where the extract is concentrated between 5 to 20 times.

11. The process according to claims 7 to 10, CHARACTERIZED in that it also comprises a subsequent lyophilization step.

12. Use of the extract according to claim 1, wherein food coloring is used to stably at temperatures between 4 ^o and -20 ° C.

13. Use of the extract according to claim 1, CHARACTERIZED in that it serves to color food in a stable manner at pH between 1 and 5.