ES2802289B2 - MEAT FOODS WITH HEALTHY PROPERTIES INCLUDING A MIX OF NATURAL BIOACTIVES - Google Patents

Description

DESCRIPTION

Meat foods with healthy properties that include a mixture of natural bioactives

TECHNICAL SECTOR

The present invention relates to a mixture of bioactive compounds of a phenolic nature from different plant extracts and to a meat product containing said composition. Therefore, the present invention falls within the field of food technology and the food industry.

BACKGROUND OF THE INVENTION

Currently, people are well aware of the importance of eating a varied and balanced diet, with nutritional aspects increasingly influencing the decision to buy certain foods. These new circumstances require going a step further and trying to provide a new range of healthy meat foods, ready for consumption at any time and in any place safely. In this sense, the article Nutr Hosp. 2014; 29 (6): 1197-1209 provides a review and analysis of the incorporation of bioactive compounds in meat products and the beneficial effects on health that the intake of these enriched products entails.

In recent years, numerous studies have endorsed the beneficial effects of polyphenol intake on health. These effects are mainly a consequence of its antioxidant properties, although they also have vasodilator and anti-inflammatory activity, in addition to having shown the ability to improve the lipid profile and attenuate the oxidation of low-density lipoproteins (LDL).

Document US2008 / 0286254 describes a composition that contains polyphenolic compounds from licorice that can be added to food and beverages and that has a physiological effect on the accumulation of fats, making it useful for the treatment of obesity or metabolic syndrome.

In the document Meat Sci. 2010; 85 (2): 274-9, a sausage formulation is described fermented that includes ingredients that contribute a nutritional improvement. These components include lemon balm extract as a source of natural antioxidants.

Taking this into account, the addition of antioxidant bioactive agents with beneficial physiological effects such as phenolic compounds from plant extracts can be very useful to obtain a meat product or any other food based on healthier meat protein with the aim of preventing different chronic diseases.

DESCRIPTION OF THE INVENTION

The present invention describes the development of a composition that is a combination of different phenolic compounds from a variety of plant extracts that are added to meat products to enrich them. The incorporation of this composition gives the meat product a series of antioxidant and anti-inflammatory properties with specific benefits to preserve the health of the end consumer. Furthermore, this composition interacts with the protein matrix to which it is incorporated in a way in which there is no loss of the organoleptic characteristics of the final product.

Therefore, in a first aspect, the present invention relates to a composition comprising the following phenolic compounds:

- between 10 and 50% by weight of chlorogenic acids,

- between 4 and 35% by weight of catechins,

- between 2 and 15% by weight of epigallocatechins,

- between 1 and 30% by weight of hydroxytyrosol.

This composition can also contain between 5 and 30% by weight of other phenolic acids, different from the previous ones.

In a preferred embodiment, the composition of the invention comprises between 30 and 45% by weight of chlorogenic acids.

In another preferred embodiment, the composition of the invention comprises between 7 and 25% by weight of catechins.

In another preferred embodiment, the composition of the invention comprises between 3 and 8% by weight of epigallocatechins.

In another preferred embodiment, the composition of the invention comprises between 4 and 25% by weight of hydroxytyrosol.

Additionally, the composition of the invention may contain other compounds of phenolic nature, such as flavones and flavanones, anthocyanins, rosmarinic acid, as well as L-ascorbic acid.

In a preferred embodiment, the composition of the invention comprises between 0.1 and 5% by weight of anthocyanins.

In another preferred embodiment, the composition of the invention comprises between 5 and 10% by weight of rosmarinic acid.

In another preferred embodiment, the composition of the invention comprises between 5 and 10% by weight of flavones and flavanones.

In another preferred embodiment, the composition of the invention comprises between 2 and 10% by weight of L-ascorbic acid.

The compounds that comprise the composition of the invention come from natural extracts of vegetable origin. In the present invention, plant extract is understood to be the products extracted directly from plants, including their different parts such as seeds, leaves, stems, roots, flowers, fruits, etc., which contain a high concentration of active principles that can perform a beneficial function in the body through a food product or cosmetic. Plant extracts can be obtained by standard chemical extraction techniques (for example, using water to extract water-soluble compounds, alcohols and acetone to extract fat-soluble compounds, or mixtures of these). The extract can then be purified to reduce the number of compounds in the extract or even isolate a single compound or class of compounds using known technologies such as chromatography and crystallization.

Preferably, the vegetable extracts from which the compounds of the composition of the present invention are obtained come from pomegranate, grape, acerola, cocoa, green tea, red tea, black tea, green coffee, olive tree, rosemary, camucamu, lemon balm or combinations thereof, and more preferably green coffee, green tea, olive, rosemary, or combinations thereof.

The compounds that form the composition of the invention include the various isomeric forms described.

The total amount of bioactive phenolic compounds present in the composition described above represents between 40 and 75% by weight of the total composition and preferably between 30 and 60% by weight of the total composition.

Another aspect of the invention relates to a meat product comprising the composition of the invention that has been described above.

Preferably, this meat product contains between 0.1 and 2% by weight of the composition of the invention with respect to the total weight of the meat product.

In the present invention, a meat product is understood as any processed product resulting from the transformation of meat, from one or more animal species, or from the new transformation of said transformed products, so that the cut surface shows that the product it has ceased to possess the physicochemical characteristics of fresh meat. Preferably, the meat product is a sausage that can be cooked, such as cooked ham or York, turkey or chicken breast, chowder, mortadella, etc .; or it can be a raw-cured and / or fermented meat product such as sausage, salami, chorizo, fuet, etc .; as well as any reduced salt and fat variety of the above.

The meat product of the invention may contain other substances with antioxidant properties compatible with the matrix, such as zinc or selenium mineral salts in any of their varieties suitable for human consumption.

Another aspect of the invention relates to the use of the composition described above for obtaining enriched meat products. The incorporation of the composition into the meat matrix can be carried out at any stage in the manufacture of the meat product.

EXAMPLES OF EMBODIMENT OF THE INVENTION

Example 1: obtaining the composition of the invention.

Various plant extracts were mixed depending on the target meat matrix. The mixtures tested were the following:

- Mixture A: composed of a combination of tea, coffee, rosemary and olive extracts, indicated for a cured and fermented raw meat product, such as salami or chorizo in a concentration of variable use and between 0.2 and 2% .

Mix A : Composition range of bioactive compounds in (%) by weight:

Chlorogenic acids: between 37 and 48%

Catechins: between 12 and 25%

Epigallocatechin: between 3 and 7%

Hydroxytyrosol: between 4 and 22%

Rosmarinic acid: between 8 and 10%

Flavones flavanones: between 5 and 7%

Other phenolic acids: between 17 and 23%

- Mix B: made up of a combination of coffee, tea, olive and acerola extracts, indicated for a cooked meat product such as ham, in a variable use concentration between 0.1 and 1%.

Mix B : Composition range of bioactive compounds in (%) by weight:

Chlorogenic acids: between 35 and 45%

Catechins: between 5 and 20%

Epigallocatechin: between 2 and 6%

Hydroxytyrosol: between 15 and 25%

Anthocyanins: between 0.5 and 5%

Other phenolic acids: between 15 and 18%

• L-ascorbic acid: between 4 and 8%

Next, we proceeded to test both the functional activity of the mixtures and the benefits of the meat products that include them, at two scales in-vitro and in vivo, using models of studies at the cellular, preclinical and intervention levels in the human population. , which are described below.

Example 2: measurement of antioxidant activity in vitro.

The ABTS + and FRAP method has been carried out to measure the antioxidant capacity of the different combinations, the results of which are shown in Table 1. The ABTS test is a method that allows evaluating the antioxidant capacity of a compound or a mixture according to the capacity reflected by these to sequester free radicals. For this, the radical ABTS + is generated, which faces the mixture of bioactive substances under evaluation.

Table 1: Antioxidant capacity (ABTS + Eq ^ Moles Trolox / 100g) of the mixture of bioactives, meat product and enriched meat product.

In the FRAP method, the antioxidant capacity is determined indirectly. It is based on the power of an antioxidant substance to reduce Fe3 + to Fe2 + which is less antioxidant. The colorless ferric-2,4,6-tripyridyl-s-triazine (TPTZ) complex is reduced to the colored ferrous complex. Because the redox potential of Fe3 + -TPTZ is comparable to that of ABTS +, similar compounds can be analyzed with both methods, although the reaction conditions are different. The mechanism of the FRAP is electron transfer, unlike other methods where free radical capture occurs, according to this, the FRAP can be useful, in combination with other methods, in determining the antioxidant capacity of products that contain different types of antioxidants.

Table 2: Antioxidant capacity (FRAP Eq pMoles Trolox / 100g) of the mixture of bioactives, meat product and enriched meat product.

Example 3: measurement of antioxidant and anti-inflammatory activity at the cellular level.

Macrophages RAW 264.7:

The RAW264.7 cell line is a type of macrophage present in the Mus musculus race (common mouse) with an induction of leukemia by a virus. The mouse strain used was BALB / c. Macrophages originate from monocytes through differentiation and are usually in a quiescent state. This rest can be altered through various stimuli that occur during the immune response. They belong to the innate immune system since these, thanks to the membrane receptors of bacterial molecules such as the lipopolysaccharide receptor CD14, can be stimulated against the presence of pathogens and produce an immune response. For example, through the LPS of Escheríchia coli O127: B8 thus producing a series of markers such as:

• Reactive oxygen species (such as superoxide anion)

• Reactive nitrogen species (such as nitric oxide radical)

• Cytokines (such as IL-1B, IFN-gamma, or TNF-alpha)

A reduction in reactive oxygen species (ROS) would imply a reduction in oxidative damage associated with an immune response.

The nitric oxide radical is produced through NO synthase by the metabolism of the amino acid arginine to citrulline, a process that is exacerbated in a stimulated macrophage. The mediation in the production of nitric oxide supposes a modulation of the inflammatory process, referring to its reduction as a reduction in the intensity of the inflammatory process and the damage associated with it. Likewise, the reduction of pro-inflammatory cytokines is indicative of a reduction in the magnitude of the inflammatory process.

The RAW264.7 cells were cultured in 96-well plates, obtaining a mature population of macrophages to which clean medium was added and waited 24 hours. Subsequently, the test solution (mixtures of extracts A and B) was added to each well (except for the positive and negative controls) and incubated for another 24 hours. The immune response was induced by adding new medium enriched with 1pg / ml of lipopolysaccharide.

After a last incubation for 24 hours, the supernatant, which mainly contains nitric oxide and cytokines, is separated from the intracellular medium in which reactive oxygen species are measured.

Table 3: Percentage of inhibition of the mixtures of bioactives of natural origin and the enriched meat products on ROS, NO and several pro-inflammatory cytokines in the RAW 264.7 macrophage cell model compared to the control stimulated with

LPS.

HL1 cardiomyocytes:

This model allows the study of different biochemical mechanisms present in the cardiomyocyte in the absence of external factors such as hormonal or inflammatory response. The HL-1 cells were subjected to a standard protocol of damage by Angiotensin II, as a model of hypertension or to a protocol of simulated ischemia due to hypoxia and absence of glucose.

The characterization of the protective effect was evaluated by means of: (1) the colorimetric analysis with MTT of the percentage of cell viability; and (2) the determination of the oxidative stress. (a) The levels of reactive oxygen species (ROS) were obtained in a spectrofluorimeter using the DCFDA fluorescence probe, which is sensitive to the presence of ROS; (b) Glutathione peroxidase and catalase antioxidant enzyme activity was determined using activity kits supplied by Enzo Life Sciences (Farmingdale, NY, USA); (c) MDA levels were determined by colorimetry according to established protocols.

The data generated in the same cell model, but with the simulated ischemia protocol and using the same meat products enriched with the bioactive mixture provided information similar to that obtained in the Angiotensin II hypertension model.

Table 4: Effect of various meat extracts supplemented with two different bioactive mixtures on catalase and GpX3 activities and levels of malon dialdehyde (MDA) and reactive oxygen substances (ROS) in HL-1 cell cultures treated with Ang-II

CCD18 myofibroblasts:

Human colon myofibroblasts (CCD18-Co cell line) were also used as a cell model. The CCD-18Co line are adherent cells with fibroblastic morphology that come from normal colon tissue. These cells reach senescence upon reaching a PDL of 42 ( population doubling level; population doubling time). All the tests have been carried out with cells whose PDL was between 32-39.

The CCD-18Co colon myofibroblasts were cultured in EMEM medium (Eagle's minimal essential medium) supplemented with 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 1.5 g / L sodium bicarbonate, 100 U / mL penicillin, 100 qg / mL streptomycin (Gibco, Invitrogen SA, Barcelona, Spain), 10% v / v of fetal bovine serum (FBS), at pH 7.2-7.4 and kept at 37 ° C with 5% CO ² and a constant relative humidity (RH) of 95%.

The anti-inflammatory effect was evaluated on a model of normal CCD18-Co colon cells subjected to an inflammatory challenge with IL-1 p (1 ng / mL). For its evaluation, the effect on the production of prostaglandins (PGE2) in response to the inflammatory stimulus was analyzed.

Table 5: Percentage of inhibition of the mixtures of bioactives of natural origin and the meat products enriched on PGE2 in the CCD18 myofibroblast cell model with respect to the control stimulated with IL-1 p.

Example 4: measurement of antioxidant and anti-inflammatory activity at the pre-clinical level.

For the pre-clinical demonstration of the potential beneficial effects of both the bioactive mixture and the enriched meat product, an animal model was used where a chronic inflammatory process could be induced and thus quantify the improvement that the direct incorporation would bring to the diet of the mixture of bioactives or through their inclusion in the reformulated meat product. This study was carried out using a population of 36 C57BL / 6 mice. This animal model in addition to being one of the most used in scientific research and therefore has an important bibliographic support, it has a high similarity with what happens in the human population, which facilitates the extrapolation of the results found in the inflammatory and oxidative processes.

Obesity was chosen as the research model since the metabolic syndrome derived from it induces a chronic inflammatory process, increasing oxidative stress and inflammatory status, making it easier to observe a potentially modulating effect on the parameters that are the object of the intervention.

With the proper authorization of the bioethics committee, the mice were divided into various intervention groups, fed ad-libitum with the following diets: (1) obesogenic control diet; (2) obesogenic diet enriched with the mixture of antioxidant bioactives (dose of 250 mg / kg / day) and (3) obesogenic diet that included 7.5 g / kg / day of the enriched meat product. All diets were normalized with the same macronutrient profile.

From the beginning to the end of the intervention period, the analysis of different biomarkers of oxidation, inflammation and immunomodulation was carried out in both urine and blood samples, in addition to the corresponding anthropometric follow-up complemented by the use of histological and SPEC imaging techniques. CT.

Urine from obese mice treated with reformulated meat product was the most antioxidant of all. Likewise, the antioxidant capacity of the plasma measured by the FRAP method, and by the levels of antioxidant enzymes PON1, GPox and catalase were increased in the groups fed with feed that included both the mixture of bioactives and the meat product enriched with said mixture. The increase seen in the antioxidant enzymes catalase and glutathione peroxidase were associated with an increase in endogenous antioxidant defenses and therefore with greater resistance to oxidative stress (catalase activity). For its part, the increase in glutathione peroxidase activity helps prevent damage derived from oxidation in DNA, proteins and lipid membranes. On the other hand, the levels of oxidized LDL were reduced in the same groups with respect to the control, the relationship between the reduction of this biomarker and the prevention of cardiovascular diseases being widely described.

Table 6: Levels of FRAP, antioxidant enzymes, pro-inflammatory cytokines and oxidized LDL in plasma in the different groups of C57BL / 6 mice.

Regarding the biomarkers of inflammation, both at the intestinal and plasma levels, important modulations were observed after the consumption of the bioactive mixture, including or not the meat matrix, the most relevant being those found at the plasma level for IL-1p, IL -10, IL-12p70, MCP-1 and TNF-a. These results indicate a systemic modulation of chronic inflammation stimulated through the induction of obesity.

Regarding the anthropometric study supported by tissue histology and the observations in SPEC-CT, it was possible to conclude that the animals that were fed with the inclusion of cooked ham enriched with the mixture of natural bioactives (phenolic compounds) reduced both the total volume of body fat and the deposition of fat in the liver. Both modifications are considered beneficial for health not only because of their direct implication in the control of body weight but also because of their importance in reducing the impact of the metabolic syndrome, which causes adipose tissue hyperplasia in obesity, and therefore in obesity. possible comorbidities derived from fatty liver

Example 5: Measurement of anti-inflammatory and immunomodulatory activity at the clinical level

The last work package presented is a nutritional intervention of short duration (4 weeks) to evaluate the possible beneficial effects that the consumption of a product reformulated with one of these mixtures has on human health. For this, a longitudinal, double-blind, placebo-controlled nutritional intervention was performed in two parallel groups. The population of both groups consisted of healthy adults (15 people / group) between 20 and 60 years old, with a body mass index between 18.5 and 25, who did not previously consume antibiotics 3 months before the intervention and did not suffer from previous pathologies (diabetes, hypertension, immunological disorders, etc.). The meat product used for the control population corresponded to a standard fermented and cured product, while the intervention group consumed the same product, but enriched with the bioactive composition described in the present invention.

The intervention lasted for 5 weeks, the first of which was washing and the remaining 4 were intervention in which each participant consumed 30g of product daily. On the first day of the intervention, the participants attended the first session on an empty stomach and a stool sample was made (taken between the night before and the morning of the intervention, determining the SCFA content, antioxidant capacity and composition of the intestinal microbiota ), a blood test (complete blood count, uric acid, liver transaminases, total cholesterol, HDL, LDL, blood glucose, CRP, MDA and oxidized LDL, plasma catalase and glutathione peroxidase, IL6, IL10, TFNa and phagocytic activity) and an anthropometry ( height, weight, BMI, bioimpedance, perimeters and folds, and blood pressure). During the 4 weeks of intervention, the participants were followed up through weekly reviews of their gastrointestinal function, response to satiety and food intake through questionnaires. At the end of the intervention, a new anthropometric, blood and stool examination was carried out.

The anthropometric analysis and the hematological parameters did not show statistically significant differences (p> 0.05) between both groups. In this way, it was possible to conclude that the intake of 30 grams of product (either improved or the control product) not only does not change the hematological parameters, but also does not alter body weight or blood pressure.

The results obtained at the biochemical level were very interesting, since on the one hand No statistically significant differences were found between the different groups, especially at the level of cholesterol and triglycerides. However, only in the group that consumed the product enriched with the mixture of antioxidant bioactives did HDL cholesterol levels tend to rise (p = 0.061) and LDL cholesterol levels to decrease (p = 0.059), inducing a clear trend to improve the lipid profile.

Regarding the plasma oxidation markers, they showed that the consumption of the reformulated product improved the antioxidant defenses (p <0.05), since the level of the antioxidant enzymes catalase and glutathione peroxidase increased, while the levels of oxidized LDL ( reflecting possible future atherosclerosis problems) decreased.

Likewise, the analysis of the plasma malondialdehyde (MDA) content, a marker of endogenous lipid peroxidation, showed a statistically significant decrease (p <0.05) after consumption of the product improved with the bioactive mixture. Finally, the levels of C-reactive protein (CRP), a nonspecific inflammatory marker, were determined, which also decreased significantly (p <0.05) in the intervention group at the postprandial level.

At the immunological level, a significant decrease (p <0.05) was found in the plasma TNFa levels of the intervention group, which indicates a clear immunomodulatory activity of the product with the bioactive mixture. Likewise, although no significant differences were found between the control group and the intervention group in the levels of IL6 and IL10, it is true that IL6 tended to decrease (p = 0.067) and those of IL10 to increase (p = 0.059) , reinforcing the potentially immunomodulatory effect of the product thus reformulated.

Finally, the intestinal health of the participants was evaluated through the production of SCFAs and the modification of the composition of the intestinal microbiota. A higher production of SCFAs (immunomodulators) was found in the intervention group (p <0.05), which further supports the levels of immonumodulation explained in the previous sections.

It is shown through the study of nutritional intervention in the human population, how the regular consumption of the meat product described in the present invention can exert a beneficial effect for the health of the consumer in the long term since:

- It has an anti-inflammatory and immunomodulatory effect.

- Improves the oxidative state, which reinforces its cardioprotective role. - Improves intestinal integrity (due to the action of butyric acid) nutritional.

Claims (21)

1. Composition comprising the following compounds: - between 10 and 50% by weight of chlorogenic acids, - between 4 and 35% by weight of catechins, - between 2 and 15% by weight of epigallocatechins - between 1 and 30% by weight of hydroxytyrosol. Composition according to the preceding claim, comprising between 30 and 45% by weight of chlorogenic acids. Composition according to any of the preceding claims, comprising between 7 and 25% by weight of catechins. Composition according to any of the preceding claims, comprising between 3 and 8% by weight of epigallocatechins. Composition according to any of the preceding claims, comprising between 4 and 25% by weight of hydroxytyrosol. Composition according to any of the preceding claims, further comprising between 0.1 and 5% by weight of anthocyanins. Composition according to any of the preceding claims, further comprising between 5 and 10% by weight of rosmarinic acid. Composition according to any of the preceding claims, further comprising between 2 and 10% by weight of L-ascorbic acid. Composition according to any of the preceding claims, further comprising between 5 and 10% by weight of flavones and flavanones. Composition according to any of the preceding claims, wherein the compounds come from plant extracts. Composition according to the preceding claim, where the plant extracts come from pomegranate, grape, acerola, cocoa, green tea, red tea, black tea, green coffee, olive tree, rosemary, camucamu, lemon balm or combinations thereof. 12. Composition according to the preceding claim, wherein the plant extracts come from green coffee, green tea, olive, rosemary or combinations thereof. Composition according to any of the preceding claims, wherein said compounds represent between 40 and 75% by weight of the total composition. Composition according to claim 13, wherein said compounds represent between 30 and 60% by weight of the total composition. 15. Meat product comprising the composition according to any of the preceding claims. 16. Meat product according to the preceding claim, wherein the composition is in a proportion of between 0.1 and 2% by weight with respect to the total weight of the meat product. 17. Meat product according to any one of claims 15 or 16, wherein the meat product is a sausage. 18. Meat product according to claim 17, wherein the sausage is cooked. 19. Meat product according to claim 17, wherein the sausage is raw cured. 20. Meat product according to claims 15 to 19, wherein the meat product has a reduced salt and fat content. 21. Meat product according to claims 15 to 20, wherein the product Meat includes mineral salts of zinc or selenium in any of its varieties suitable for human consumption.