- Analytical chemistry is an essential qualitative and quantitative tool to describe foods and biomaterials and their changes during production, processing and distribution. In this work, rapid analytical procedures for the extraction and quantification of components in fluid milk and diatom fermentation broth were developed. Analytical methods to measure the concentration of vitamin A, E & C in fluid milk were developed and applied to quantify these vitamins in milk distributed commercially in Spain and their retention in milk subjected to pressure-assisted thermal processing (PATP). Analytical procedures were also developed for the quantification, recovery and purification of chitin in diatom photobioreactor broths
and for its conversion into glucosamine by HCl acid hydrolysis. This information was required for a techno-economical and life-cycle analysis of diatom-produced chitin for food and biomedical applications, and of its conversion into glucosamine to be marketed as a nutraceutical. This analytical development work lead to the following five studies.
In the first study, total ascorbic acid (AA) in whole/semi-skim/skim raw/pasteurized/UHT milk packaged in opaque bags, transparent plastic, cardboard, and Tetra Brik™ and collected from processing plants and retail outlets was extracted and quantified by HPLC using a C-18 column. AA content ranged 0.21-10 and 3.4-16 mg/l in milk from retail outlets and processing plants, respectively, and was higher in organic milk. For same processor/lot samples, pasteurized milk showed higher AA content than UHT milk but this was not true for retail outlets samples. Among UHT samples, the AA content trend was whole<semi-skim<skim and lower for UHT milk in opaque plastic and Tetra Brik™ containers. In milk stored 14 d at 4ºC in the dark, AA losses ranged 35-83% depending on milk type. AA retention was higher in unopened milk containers.
In the second study, an extraction and HPLC quantification method was improved to allow the simultaneous quantitation of retinol, α-tocopherol, α-tocotrienol and β-carotene in commercial whole/semi-skim/skim samples of raw/pasteurized/UHT milk commercialized in transparent plastic/glass bottles and Tetra BrikTM containers. The fat-soluble vitamin content in raw, pasteurized conventional/organic, and UHT milk ranged 0.055-5.540 (retinol), 0.135-1.410 (α-tocopherol), and 0.040-0.850 mg/l (β-carotene). No significant differences (p>0.05) were observed on the losses of retinol, α-tocopherol, and β-carotene content in UHT whole milk after 5 d at 4ºC in the dark. After 14 d at 4ºC in the dark the contents of retinol, α-tocopherol, and β-carotene remained higher in milk with higher fat content and were higher in unopened milk containers. In UHT whole milk samples containing 0.02% NaN₃, retinol (33%) and αtocopherol (11%) but not β-carotene (2%) decreased significantly (p<0.05).
The third study focused on the effect of elevated pressure (200-705 MPa) at moderate temperatures (30-75º) on the AA, vitamin A (retinol, β-carotene), and vitamin E (α-tocopherol) retention in raw-whole and pasteurized-skim milk. This study showed minimum losses of retinol, α-tocopherol and β-carotene after high pressure treatments at moderate temperatures. However, even the least severe treatment (400 MPa/31°C/5 min) resulted in a statistically significant AA loss (p<0.05) in raw whole (20%) and pasteurized skim milk (13%) when compared with untreated controls. AA losses reached a maximum loss of 55 and 68% in raw whole and pasteurized skim milk, respectively, when treated for 5 min at 705 MPa and 72°C.
The focus of the fourth study was the determination of the chitin amount present in fermentation broths produced in a diatom photobioreactor. This was achieved by acid hydrolysis conversion of chitin into glucosamine quantified by HPLC. This information was essential for a techno-economical and life-cycle analysis of the chitin production by diatom cells and its subsequent conversion into glucosamine. Diatoms are single-cell algae with intricately structured cell walls made of nanopatterned silica (SiO₂) previously studied for applications in nanotechnology. This study focused on the long chitin fibers extruded by Cyclotella spp. diatoms to be used as is for biomedical and food applications or it can be hydrolyzed into glucosamine to be marketed as a dietary supplement. Kinetics of the glucosamine (GlcN) monomer production by acid hydrolysis of commercial diatom chitin was determined in 4M, 6M, 8M, 10M and 12M HCl at 90°C for up to 3 h. The GlcN produced was quantified by HPLC (Dionex MA-1 column, 30°C, 0.4 ml/min 0.75M NaOH isocratic mobile phase, ED-40 PAD detector). Acid hydrolysis in 8M HCl at 90°C for 2 h of commercial chitin suspended in distilled water showed a high GlcN conversion (98.0% ± 0.04, n=2). Tests using reagent-quality GlcN showed that the chitin monomer was stable under these hydrolysis conditions (>88% retention). However, the 8M HCl hydrolysis of commercial diatom chitin suspended in two different formulations of sterile diatom fermentation broths for 1, 2 and 3 h showed that the chitin conversion to glucosamine was fermentation media dependent. Further tests of chitin produced by diatoms in the photobioreactor confirmed that the acid hydrolysis of chitin is media dependent. To overcome this media-dependence limitation, diatom photobioreactor broth samples corresponding to initial, mid and final fermentation time points were spiked to obtain an average media correction factor. This factor was determined for each photobioreactor run to quantify chitin production using glucosamine yield after 8M HCl hydrolysis at 90°C for 3h. The correction factor allowed reliable and low-variability determinations of the chitin production kinetics in the diatom photobioreactor.
The fifth study focused on the development of a chitin separation and purification method from the diatom photobioreactor broth. A mild centrifugation step to remove diatom cells in the pellet was optimized (1500g, 1 min) and yielded a high retention of chitin in the supernatant (96.6±0.18 and 84.6±0.02) when evaluated using two different fermentation broth formulations. Intense centrifugation (11000g, 30 and 60 min) was then used to recover chitin fibers in a second pellet. Analysis of the chitin content in the supernatant of the second centrifugation showed losses of less than 10%. Therefore, the two centrifugation steps allowed a recovery exceeding 80%. The chitin pellet was then purified using 1M HCl at 70°C to solubilize calcium and other salts, 0.5% w/w SDS to remove insoluble proteins, and 95% ethanol to remove chlorophylls and other organic materials yielding chitin with a 70.9±16.6% purity.