Adipic acid

For the synthesis of the oligoesters, the following materials were used: Lipase B from Candida antarctica (CaLB, EC 3.1.1.3, specific enzymatic activity = 368 TBU/g, immobilized on epoxy-acrylic resins), 1,4-butanediol (CAS No. 110-63-4, purity = 99%), glycerol (CAS No. 56-81-5, purity = 99,5%), adipic acid (CAS No. 124-04-9, purity > 99%), dichloromethane (CAS No. 75-09-2, purity > 99.9%), hydrogen peroxide solution (CAS No. 7722-84-1, concentration = 30%), deuterated chloroform (CAS No. 865-49-6, purity = 99.8%), oleic acid (CAS No. 112-80-1, purity = 99%), linoleic acid (CAS No. 60-33-3, purity = 99%), and linolenic acid (CAS No. 463-40-1, purity = 70%) were supplied by Sigma-Aldrich (Milano, Italy)-). Azelaic acid (CAS No. 123-99-9, purity = 98%) and cardoon oil, with iodine values of 125 (gI₂/100gsample), were kindly donated by Novamont S.p.A. (Novara, Italy). Hydrogen peroxide (60 wt.%) was kindly provided by Solvay Italia (Bollate, Italy). All of the other chemicals were purchased from Merck and Sigma (Milano, Italy) (analytical or reagent grade), and they were used as received.

A total of six Pixian broad-bean paste samples were obtained from Chengdu, including sample 1 (Sichuan Province Dandan Condiment Co. Ltd., Chengdu, China), sample 2 (Sichuan Pixiandouban Co. Ltd., Chengdu, China), sample 3 (Sichuan Gaofuji Food Co. Ltd., Chengdu, China), sample 4 (Chuanjiao Branch Co. of Sichuan Youlian Seasoning and Foods Co. Ltd., Chengdu, China), sample 5 (Sichuan Chuanpidouban Co. Ltd., Chengdu, China) and sample 6 (Chengdu Wangfeng Food Co. Ltd., Chengdu, China). Fermentation period of all Pixian broad-bean paste samples was near one year. The samples were ground and stored at 4 °C until analysis.
All chemicals used in this study were of the analytical and chromatographic grade. Standard organic acids (oxalic acid, tartaric acid, citric acid, lactic acid) were purchased from Kelong chemical plant (Chengdu, China). Other standards such as succinic acid, ketoglutaric acid, fumaric acid and adipic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA), while malic acid was purchased from Aladdin Bio-Chem Technology Co. Ltd. (Shanghai, China). All amino acid standards were obtained from Seebio Biotechnology Co. Ltd. (Shanghai, China). For amino acid analysis in the automatic amino acid analyzer, ion exchange column was pre-standardized using all amino acid standards.

Samples ranging from 0.01 to 500 mg of PHB-containing material were digested in 1 mL of concentrated sulfuric acid at 90 °C for 30 min. The tubes were cooled on ice, and 4 mL of 0.014 N H₂SO₄ were added under rapid mixing. Before HPLC analysis, the samples were diluted 5 to 100 times with 0.014 N H₂SO₄ containing 0.8 mg of adipic acid (Sigma) per mL as an internal standard; they were then filtered through a membrane filter with a pore size of 0.45 µm (HAWP, Millipore Corp) to remove particulate material. The injection volumes ranged from 10 to 50 µL and the sample concentrations from 0.2 to 560 µg/mL. Samples were eluted with 0.014 N H₂SO₄ at a 0.7 mL/min flow rate from an Aminex HPX-87H ion exclusion organic acid analysis column (C18 4.6 × 250) (Torrance, CA, USA) preceded by an Aminex HPX-85X ion exclusion guard column. HPLC analysis was performed using either a Waters Associates 6000A solvent delivery system with a U6K injector, or a series of three chromatographs (PerkinElmer, Inc., Norwalk, Conn.) with a variable loop injector. The absorbance of crotonic acid was measured at 214 nm (Waters 441 absorbance spectrophotometer) or 210 nm spectrophotometer. The amount of crotonic acid produced from PHB was calculated by using the regression equation derived from known crotonic acid standards [25] (link).

Glycolic acid, dimethyl
terephthalate, adipic acid, succinic acid, bismuth(III) subsalicylate,
antimony(III) oxide, zinc acetylacetonate, zinc acetate, methanesulfonic
acid, phosphoric acid, DMSO-d₆, and CDCl₃ were purchased from Sigma-Aldrich. 1,4-Butanediol,
tin(II) 2-ethylhexanoate, and titanium butoxide were purchased from
VWR, Alfa Aesar. Zirconium(IV) acetylacetonate was purchased from
Strem Chemicals U.K., Ltd. Irgafos 126, Irgafos 168, and Joncryl ADR
4468 were from BASF.

Tributylamine (99%), trihexylamine (99%), trioctylamine (99%), dimethyl carbonate (99%), adipic acid (99%), fumaric acid (99%), tartaric acid (99%), salicylic acid (99%), hexanoic acid (99%), sodium acetate (99%), heptane (98%), ethanol (99%), and methanol (99%) were purchased from Sigma Aldrich and used as received. Deionized water was acquired from Laboratorio Ambiental CIITEC-IPN; commercial beetroot extract with 12% of betanins (CBE) and beetroot were bought at a local market.
All the synthesized compounds were characterized by ¹H and ¹³C NMR and infrared (IR) and ultraviolet–visible (UV-Vis) spectroscopic techniques. IR/UV-Vis spectra were recorded on a Thermo Scientific Nicolet 8700 spectrometer. ¹H (300 MHz) and ¹³C NMR (75.40 MHz) spectra were recorded on a JEOL Eclipse-300 in CDCl₃, and chemical shifts were expressed in ppm with tetramethylsilane as the internal standard.