Current interest in health has led to an increase in demand for functional food supplements as well as in industry concern for maintaining the bioactive compounds of such foods via the application of new technologies. In this study, we evaluated the effect of moderate high-pressure homogenization (HPH) treatments (80 and 120 MPa) versus thermal treatment (80°C, atmospheric pressure) on the functional bioactive compounds from four different functional supplements stored under accelerated conditions (40°C ± 2°C and 75% ± 5% relative humidity) for 6 months. HPH proved to be a better alternative than thermal treatment for functional supplements containing heat-sensitive compounds such as vitamin C, vitamin A, and unsaturated fatty acids (10-hydroxy-2-decenoic acid). The proanthocyanidin, cynarin, chlorogenic, and iron contents, however, were not initially affected by HPH treatments. The storage time caused important reductions in the majority of the compounds studied (mainly in vitamins C, B12, and A), although the lowest decrease was found in the HPH samples. The food matrix had an important effect on the final functional composition and required the optimization of HPH treatments for each functional food supplement. HPH is a recommended alternative to thermal treatment for functional food supplements, in particular when they are rich in thermolabile bioactive compounds.
The current trend in developed societies towards an unhealthy lifestyle results in factors such as stress, unbalanced diets and unhealthy eating habits, a lack of physical exercise, and many other factors that have a negative impact on our health. In addition, a number of diseases and physiological conditions (colon cancer, gastrointestinal conditions, and pregnancy) can produce iron deficiency, which is the most common and widespread nutritional deficiency worldwide that can lead to anemia [
Nevertheless, functional food supplements have to be manufactured and then stored in proper conditions in order to be maximally effective. Along these lines, the use of novel technologies such as high pressure could help reduce the degradation of such bioactive compounds. There are two different high-pressure technologies depending on the application technique, although both have some common features. The first one, known as high hydrostatic pressure (HHP), uses water as the pressure transmitting medium and subjects liquid and solid foods to 100–1000 MPa at room or mild processing temperatures. This technique instantaneously transmits isostatic pressure to the product, independent of its size, shape, and food composition, yielding highly homogeneous products [
There are numerous reports describing the effect of HHP on the bioactive content of fruit juice and purée [
To prepare the functional supplements, the following ingredients were used: dry cranberry (
Four functional supplements with different functional purposes were made under Clean Room conditions (Class 10,000). One product made from cranberry juice extract rich in proanthocyanidins was targeted for cystitis treatment. The second functional product made from dry artichoke extracts was targeted for liver diseases. This product contained cynarin and other caffeoylquinic derivatives such as chlorogenic acid. The third product was made from royal jelly with a guaranteed minimum of 1.5% 10-HDA. The final functional product was an iron supplement for the prevention of iron deficiency anemia. The composition of these four supplements (cranberry, artichoke, royal jelly, and iron) with the addition of different vitamins is shown in Table
Composition of the functional supplements studied.
Product | Main compounds | Concentration per mL |
---|---|---|
Cranberry supplement | Cranberry dry extract juice |
720 mg |
Vitamin C | 7.5 mg | |
|
||
Artichoke supplement | Artichoke dry extract |
15 mg |
Lemon juice | 0.07 mg | |
Vitamin E | 2 mg | |
Vitamin B12 | 0.25 |
|
|
||
Royal jelly supplement | Royal jelly |
15 mg |
Vitamin C | 9 mg | |
Vitamin B12 | 0.25 |
|
Vitamin A | 190 |
|
Vitamin E | 1.6 mg | |
|
||
Iron supplement | Iron | 1.5 mg |
Vitamin C | 7.5 mg | |
Vitamin B12 | 0.03 |
|
Vitamin A | 58 |
|
Vitamin E | 0.54 mg |
The control pasteurization process for each functional food supplement was prepared as described below. In a heat exchanger (Inoxpaser, RP-13/006 model, Molina de Segura 129, Spain) at 0.1 MPa (atmospheric pressure), deionized hot water (110°C) was stirred at 20 rpm in a tank. When the temperature decreased to 80°C, plant extracts were added, with this process taking 15 min. After that, at 60°C, the liposoluble compounds were added and the temperature was maintained for 5 min, and at 40°C, nonliposoluble compounds and potassium sorbate were added (1.8 mg mL−1, pH = 4.5 and 5.5). This last step took 5 more minutes. Samples were cooled until they reached 40°C and directly packed. For the HPH treatments, the same ingredients used in the control pasteurisation process were added in the same order to a tank at room temperature (22°C). In this case, and depending on the ingredient’s solubility, it was not necessary to wait until the total dilution of each one. The pressure used in the HPH treatment works by helping to emulsify the mixture between lipo- and nonliposoluble compounds, thereby reducing the processing time from the 25 min needed for the pasteurization process to less than 10 min. The mixture was kept under continuous stirring. When each food supplement had all the functional ingredients, the mixture was processed using an instantaneous high-pressure homogenizer (GEA Niro Soavi, Ariete NS3006 Model, Parma, Italy) at a flow rate of 60 L h−1. Two HPH doses were studied, 80 MPa with a final temperature after HPH treatment of 33°C and 120 MPa where samples reached 43°C. The inlet temperature and pressure for both HPH treatments were room temperature and atmospheric pressure (0.1 MPa). The treatment time was 5 seconds for each treatment. After HPH treatments, samples were collected into a tank and the homogenized mixtures of each food supplement were directly packed.
Samples from all treatments were then bottled in sterile brown glass flasks (250 mL) under nitrogen gas and sealed with polyethylene caps. The full experiment was independently conducted three times, each time constituting a repetition. All samples were stored for up to 6 months under accelerated conditions (40°C ± 2°C and 75 ± 5% RH conditions) [
The detection of ascorbic acid in the samples was monitored by HPLC. A volume of 15 mL of sample was added to 10 mL of ultrapure water and shaken for 1 min on a vortex shaker (Reax, Heidolph, Schwabach, Germany). The sample was filtered through 0.45
Determination of vitamin B12 was performed according to Campos-Giménez et al. [
Both fat soluble vitamins were analyzed according to Klejdus et al. [
The cranberry food supplement was shaken for 2 min on a vortex shaker (Reax, Heidolph, Schwabach, Germany). Then, 10 mL of this sample was centrifuged at 5,000 rpm for 10 min. A total of 1 mL of this supernatant was diluted with 0.5 mL of methanol and then filtered (0.2
Cynarin and chlorogenic acid determination was performed as described below. Samples (5 mL) were added to 5 mL of pure methanol and homogenized for 1 min. The samples were then filtered through 0.22
The royal jelly food supplement was shaken for 2 min on a vortex shaker (Reax, Heidolph, Schwabach, Germany). Then, 10 mL of the sample was acidified with 1 N HCl to pH 2.5–3.0. An aliquot of 1 mL was slowly filtered with the help of a vacuum pump through a column (Strata C18-E, Phenomenex, Madrid, Spain), which was previously activated with 3 mL of HPLC grade methanol. Then, 3 mL of ultrapure water was added. Finally, 1 mL of the sample was slowly filtered through the column to dry it. Finally, the sample was dissolved in 5 mL of methanol and filtered through a 0.2
Samples from iron food supplement were injected into an inductively coupled plasma mass spectrometer (ICP-MS, Agilent Technologies 7500 ce, Japan), where samples were vaporized and ionized by argon plasma. The iron isotope 56 was determined and quantified by comparison with an external standard. For the extraction, three replicates of samples (0.1 mL) were placed into plastic tubes, 10 mL of water was added, and serial dilution was performed until a dilution of 1 : 1000 was reached.
A randomized design with three replicates per treatment was used. To determine the effect of HPH on each sampling time of accelerated storage, a one-way analysis of variance (ANOVA,
Some parts of the discussion have been focused on HHP treatments, as there is currently a lack of information regarding the effect of HPH on functional supplements.
Before processing, the initial ascorbic acid content was 7.5 mg mL−1 in the cranberry food supplement, 9 mg mL−1 in the royal jelly, and 7.5 mg mL−1 in the iron food supplement (Figure
The effect of HPH treatments on the ascorbic acid content of cranberry (a), royal jelly (b), and iron food supplements (c) stored at 40°C ± 2°C and 75% ± 5% RH. Mean (
The level of ascorbic acid degradation was slightly different for each functional compound, depending on the functional compound’s matrix. This could be due to the fact that the ingredients in the food matrix can have influence on the bioactive compounds’ activity, degradation, or release. For instance, the application of HPH treatment (20–100 MPa) to mandarin juice with trehalose addition (10–30%) degraded the vitamin C content between 2 and 4% during storage, but this trehalose addition resulted in less flavonoid degradation during storage [
Storage under accelerated conditions strongly reduced the ascorbic acid content in all food supplements, although the remaining vitamin C in both HPH treatments was significantly higher (
Before processing, the initial vitamin B12 content in both artichoke and royal jelly supplements was 0.25
The effect of HPH treatments on the total vitamin B12 content of artichoke (a), royal jelly (b), and iron food supplements (c) stored at 40°C ± 2°C and 75% ± 5% RH. Mean (
Before processing, the initial vitamin A content was 190
Effect of HPH treatments on the total vitamin A content of royal jelly (a) and iron food supplements (b) stored at 40°C ± 2°C and 75% ± 5% RH. Mean (
Before processing, the initial vitamin E content was 2 mg mL−1 in the artichoke supplement, 1.6 mg mL−1 in royal jelly, and 0.54 mg mL−1 in the iron food supplement (Figure
Effect of HPH treatments on the total vitamin E content of artichoke (a), royal jelly (b), and iron food supplements (c) stored at 40°C ± 2°C and 75% ± 5% RH. Mean (
The time of storage led to a decrease in vitamin E, but this liposoluble vitamin was much more stable than the rest of the analyzed vitamins particularly in the royal jelly food supplement, where final losses ranged from 18 to 24% with significant differences observed between treatments. In all food supplements, samples treated under HPH had the best vitamin E retention with storage time. It is interesting to note that, in royal jelly, vitamin E showed greater stability with storage time than vitamin A (Figure
The initial proanthocyanidin content present in the cranberry dry extract used was 5.50 mg mL−1 (Figure
Effect of HPH treatments on the phenol content of cranberry (a) and artichoke food supplements (b, c) stored at 40°C ± 2°C and 75% ± 5% RH. Mean (
The polyphenols studied in the artichoke food supplement were cynarin and chlorogenic acid. Both polyphenols were naturally present in the artichoke dry extract. The initial cynarin content was 0.12 mg mL−1 with small reductions (1 to 2.5%) after thermal and HPH treatment. This compound was well preserved until the end of storage, although there were significantly higher losses under thermal (18%) than under HPH treatments (9%) (Figure
Before processing, the initial 10-HDA content in the royal jelly supplement was 2.50
Effect of HPH treatments on the 10-HDA content of the royal jelly food supplement stored at 40°C ± 2°C and 75% ± 5% RH. Mean (
The iron levels in the supplements studied ranged from 1.3 to 1.5 mg mL−1, and no significant differences were found between thermal and HPH treatments at any length of storage time (data not shown). The storage losses were around 16% in all samples, which indicated that this mineral was quite stable as compared to the vitamins studied. Recently, Hemery et al. [
HPH is a recommended alternative to thermal treatments for functional supplements containing heat-sensitive compounds. HPH also showed a positive effect on vitamins B12 and E, but this effect depended on the functional matrix. The optimization of HPH conditions depended on the bioactive compounds and functional food supplements.
The authors declare that they have no conflicts of interest.
The authors are grateful to the Development Agency of the Region of Murcia for financing part of this study. Ascensión Martínez-Sánchez holds a postdoctoral grant (“Juan de la Cierva”) from the Spanish Ministry (MINECO).