Health4Cardio EE-LV00035
Period: 01.10.2023-30.09.2025
Programme priority: Jointly and smartly growing businesses
Specific objective: 2.1: Enhancing sustainable growth and competitiveness of SMEs and job creation in SMEs, including by productive investments
Summary
Health4Cardio is a cross-border cooperation project between BioCC OÜ (Estonia) and SISTEMU INOVACIJAS JSC – Si Biotech (Latvia). The project’s focus is to use cross-border knowledge transfer to improve the partners’ capacity to develop a prototype of an innovative new dietary supplement. The partners have their own individual strengths and expertise in production and distribution. BioCC works with high qualitative lactic acid bacteria cultures and is competent in conducting clinical trials with probiotics and functional foods, whereas Si Biotech is specialized in the extraction of natural substances using modern and green extraction methods, (CO2 supercritical fluid extraction) both for research and production. The common challenge is to find a technological solution to combine a clinically researched and patented probiotic bacteria strain with sea-buckthorn (Hippiophae rhamnoides) seed oil and find a suitable delivery form to determine health benefits for cardiovascular- and gut health.
Objectives
- Finding an innovative technology for the combination of two health-beneficial ingredients to develop a synergistic dietary supplement prototype using the in-house competencies of both partners.
- Determining the prototype’s mechanism of action and bioavailability by conducting a preliminary clinical trial.
Results
Completing these actions will create innovative technologies through sharing of knowledge and consequential product development. A jointly developed technology will raise both partners’ competitiveness and growth thanks to cross-border cooperation, knowledge transfer, and innovation.
Total budget: 577 375 €
Financed by the European Regional Development Fund (ERDF): 461 900€
Co-financed by the partners: 115 475€
Contact:
Liina Kuus
liina.kuus@biocc.ee
Report Period: 01.02.2024-31.05.2024
Work package 1
During reporting period Project activity – A1.1 “Product prototype development” was complated and A1.2 “Experimental production series and scale-up of production technology” was started.
Accordingly following activities where performed:
- review of scientific and technical literature on lipid and probiotic characteristics, properties and compatibility in homogenous solution;
- design of experimental approaches on how to successfully combine lipids with probiotics using mechanical and chemical technics;
- viability, compatibility and biological activity testing;
- specification of the new product in development;
- experimental production series simulation.
Another batch of si biotech sea buckthorn seed lipid extract (SBSLE) was produced exclusively for further developments in the scope of the Health4Cardio project. SBSLE was produced by supercritical fluid extraction with CO2, according to the company’s standard extraction procedure.
In order to develop a stable and biologically active combination of lipid extract with probiotics, it was decided to perform a gentle refinement procedure to purify SBSLE from free fatty acids, phospholipids, pigments, trace elements and oxidation products, which could reduce the viability of probiotics.
The leading partner provided its active ingredient for the combination – a patented probiotic strain, Lactobacillus plantarum Inducia, weight 200g, with a bacterial count of 5 billion CFU per gram.
After reviewing scientific and technical literature and analysing existing products in the market, considering Period 1 findings and conclusions, it was decided that the jointly developed product will contain 1000 mg of active ingredients—SBSLE, bacteria, and emulsifiers. The planned delivery form is a soft-shell capsule, the shell of which will consist of either fish gelatine or plant-based material.
To ensure repeatability, three separate attempts at experimental production were performed. Prepared samples were transferred to 50mL lab tubes. One set was stored at +4°C, the second in a dark, dry place at room temperature, and the third set was sent to leading partner BioCC for viability testing.
The combination’s homogeneity, sedimentation, and precipitation will be monitored, and adjustments in product specification will be made.
Total budget: 577 375€
Financed by the European Regional Development Fund (ERDF): 461 900€
Co-financed by the partners: 115 475€
Report No.3 project “Health4Cardio”, EE-LV00035, sibiotech
Report Period: 01.06.2024-30.09.2024
During report period (Period 3) Activity A1.2 – “Experimental production series and scale-up of production technology” was implemented as wel as Activity A1.3 – “Newly developed product safety and efficiency assessment” was started. Following tasks where performed:
- The optimisation of advanced technological methodologies for the refinement of sea buckthorn seed lipid extract.
- The evolution of the most effective technological strategies for ensuring ingredient compatibility, stability, and preservation of biological activity.
- The assessment of probiotics viability within homogenised suspensions.
- A comprehensive analysis of the fatty acid profile in homogenised suspensions.
- The development and scaling of an optimised manufacturing process.
Besides, following experimental tasks where performed:
sibiotech sea buckthorn seed lipid extract (OHS01) was obtained according to the protocols outlined in WP2. The refinement process was further enhanced through modifications, wherein the lipid extract underwent advanced filtration, sedimentation, and bleaching techniques. These adjustments were designed to optimise the fatty acid composition while minimising oxidative degradation of the oil.
The purified sea buckthorn seed lipid extract was then homogenised with soy lecithin and Lactobacillus plantarum, using five distinct emulsifier concentrations, along with varied durations and rotational speeds (RPM) during the homogenisation process.
- Filtration procedure: the oil was initially heated to +50°C on a magnetic stirrer under constant rotation. It was then gradually cooled before being stored at +5°C for 24 hours. The filtration process extended over five days, with a progressive reduction in filter pore size: 25 µm on day 1, 15 µm on day 2, and 5 µm from day 3 to day 5 (Figure 1A).
- Sedimentation procedure: ethanol (96%) was added to the filtered lipid extract in a solvent-to-oil ratio of 1:2 and mixed for 30 minutes under vacuum conditions at +30°C. The resulting mixture was then transferred to separation funnels and allowed to separate for 24 hours at +5°C (Figure 1B). The lower phase, containing the lipid extract, was carefully collected, and the residual solvent was removed via rotary evaporation, following standard protocol (+40°C, 130 RPM).
- Bleaching procedure: activated bleaching earth clay, at a concentration of 1%, was added to the evaporated lipid extract. The mixture was subjected to vacuum conditions, stirred at 100 RPM, and heated to +70°C for 30 minutes. Subsequently, the mixture was filtered twice using 5 µm filters to collect the purified oil fraction.
Figure 1. Oil purifying process. A – filtration of the oil; B – sedimentation of the oil.
The chemical content analysis of the bleached oil (Table 1) revealed that the purification method effectively reduced the concentration of free fatty acids (FFA), which are stored in triglycerides (GCL). Since free fatty acids are more prone to oxidation, their reduction significantly enhances the oil’s oxidative stability, helping to prevent rancidity and maintain the oil’s quality.
Table 1.
Fatty acid composition of the bleached oil
| Fatty acid | GCL mg/g | FFA mg/g | Total mg/g | % |
| Palmitoleic acid C16:1n-7 | 6,5 | 0,7 | 7,2 | 1,05% |
| Palmitic acid C16:0 | 59,4 | 2,7 | 62,1 | 9,08% |
| Linoleic acid C18:2n-6 | 275,9 | 5,7 | 281,6 | 41,17% |
| Oleic acid C18:1n-9 + Linolenic acid C18:3n-3 | 299,1 | 4,8 | 303,9 | 44,43% |
| Stearic acid C18:0 | 28,1 | 1,1 | 29,2 | 4,27% |
| TOTAL, mg/g | 669 | 15 | 684 | 100,00% |
4. Preparation of the emulsifier: in contrast to the method outlined in WP2, soy lecithin was used as an emulsifier to achieve a homogeneous mixture. A small quantity of lecithin was incorporated into the purified oil, and the mixture was heated to +40°C under constant stirring until the lecithin fully dissolved (Figures 2A and 2B). Five samples with varying lecithin concentrations were then prepared (Figure 2C). Following this, L. plantarum was added into the oil-lecithin mixture.
Figure 2. Lecithin-oil mixture preparation. A – pure oil; B – oil with lecithin; C – five samples prepared.
The recommended concentration of lecithin 0.1% – 0.3%; samples:
- 5 x 109 CFU* + 100 g of lipid extract + 0.1% soy lecithin
- 5 x 109 CFU* + 100 g of lipid extract + 0.5% soy lecithin
- 5 x 109 CFU* + 100 g of lipid extract + 1.0% soy lecithin
- 5 x 109 CFU* + 100 g of lipid extract + 1.5% soy lecithin
- 5 x 109 CFU* + 100 g of lipid extract + 2.0% soy lecithin
*5 x 109 CFU +20% in order to confirm final dosage contains 5 x 109 CFU live Lactobacillus plantarum
5. Homogenisation procedure: a high-shear mixer was used to mix the oil and bacteria, following the methodology outlined in WP2 (Figures 3A, 3B and 3C). However, the samples were subjected to five varying homogenisation times and rotational speeds (RPM) to optimise the process:
- A. 5000 RPM / 2 min
- B. 5000 RPM / 3 min
- C. 7500 RPM / 2 min
- D. 7500 RPM / 3 min
- E. 7500 RPM / 5 min
Figure 3. High-shear homogenisation process. A – L. plantarum added to the sample; B – High-shear homogenisation; C – vacuuming process of the sample.
The samples were collected (Figure 4) and stored for future analysis of the bacteria viability and stability tests.
Figure 4. Samples for the further analysis.
6. Scale-up process: the homogenisation process was scaled up to handle 500 g of lipid extract, using 2.0% soy lecithin and high-shear homogenisation at 10 000 RPM for 7 minutes (Figure 5A). The vacuuming step was conducted using an advanced evaporation system (Figure 5B). The resulting sample was then stored for subsequent analysis (Figure 5C).
Figure 5. Scale-up process. A – high-shear homogenisation; B – vacuuming process; C – the sample of 500 g of the suspension.
Following the successful scale-up to 500 g of raw material, the process was further expanded to 5000 g (Figure 6) in preparation for trial batch manufacturing aimed at product encapsulation in soft-shell capsules. The initial product specification has been established, and a comprehensive documentation package, including the MSDS, product specification, encapsulation specification, and Certificate of Analysis (CofA), has been compiled to facilitate the ordering of the trial encapsulation batch (Table 2).
Table 2.
Specification of the product
| Product: | Sea buckthorn seed oil (scCO2) with Lactobacillus plantarum |
| Appearance: | Oblong transparent soft gelatine capsule |
| Average fill weight: | 1000,00 mg ± % |
| Best before: | |
| Ingredients of the capsule filling | |
| Capsule fill total amount | 1000,00 mg |
| Sea buckthorn seed oil (scCO2) | 988,00 mg |
| Microencapsuled Lactobacillus plantarum (5×1011 CFU/g) | 10,00 mg |
| Sunflower lecithin | 2,00 mg |
| Trace amounts of: Calcium chloride, sodium alginate. | |
Figure 6. Equipment for trial batch manufacturing, including refinement – neutralisation, bleaching and filtration, vacuum homogenisation oil with bacteria using an emulsifier.
Contact:
Liina Kuus
liina.kuus@biocc.ee