Evolution of in vitro digestibility techniques: a systematic review

The inability to reproduce certain digestive processes in vivo, high research costs and ethical aspects have led to the development of a large number of in vitro digestion models. These models allow us to take into account various factors of modeling complex multistage physiological processes occurring in the gastrointestinal tract, which makes them promising and widely used. A significant part of in vitro methods includes assessment by enzymatic digestion and are based on the calculation of nitrogen remaining after digestion in relation to the initial total nitrogen (according to the Dumas, Kjeldahl method, spectrophotometric or chromatographic method). There are also a number of titrometric methods (pH‑stat), which are mainly used to assess the digestibility of feed, most successfully for aquatic animals due to the simplicity of their digestive tract. Methods for assessing the digestibility of food products by enzymatic digestion have undergone various stages of evolution (since 1947) and have been widely modified by including various enzymes (pepsin, trypsin, pancreatin, erepsin, etc.) in model systems, indices for various products have been determined on their basis (pepsin-digest-residue (PDR) index, 1956; pepsin pancreatin digest (PPD) index, 1964; pepsin digest dialysate (PDD), 1989). As a result, a single protocol was formed to study the digestibility of food — INFOGEST (2014–2019), which includes three stages of digestion (oral, gastric and intestinal). It allows researchers to accurately reproduce the conditions of the human gastrointestinal tract and is widely used by scientists around the world.

Melting, Crystallization, and In Vitro Digestion Properties of Fats Containing Stearoyl-Rich Triacylglycerols

Fats containing the stearoyl-rich triacylglycerols (TAGs) of 1,2-distearoyl-3-oleoylglycerol (SSO) and 1,3-dioleoyl-2-stearoylglycerol (OSO) were synthesized via the lipase-catalyzed acidolysis of tristearin (SSS)-rich fat and oleic acids, followed by solvent fractionation. Their physicochemical properties and in vitro digestibilities were compared. The SSS-, SSO-, and OSO-rich fats comprised 81.6%, 52.9%, and 33.1% stearic acid, respectively, whereas oleic acid comprised 2.9%, 37.5%, and 56.2%, respectively. The SSS-, SSO-, and OSO-rich fats contained the TAGs of SaSaSa (100.00%), SaSaMo (86.98%), and MoSaMo (67.12%), respectively, and the major TAGs were SSS, SSO, and OSO, respectively. Melting and crystallization temperatures were higher and fat crystals were larger and densely packed in the descending order of SSS-, SSO and OSO-rich fats. Both in vitro multi-step digestion and pH-stat digestion were more rapid for OSO- than SSO-rich fat. Oleic acid was digested faster than stearic acid during the initial digestion, then the rate decreased, whereas that of stearic acid increased over prolonged digestion. Fats that were richer in stearoyl at the sn-1,3 position of TAG melted and crystallized at higher temperatures, had a densely packed microstructure of large fat crystals and were poorly digested. Stearic acid imparts the essential physical attributes of melting and crystallization in solid fats, and the low digestible stearoyl-rich fat would be a viable substitute for trans fatty acids in food lipid industry.

Production of R- and S-1,2-propanediol in engineered Lactococcus lactis

Abstract1,2-propanediol (1,2-PDO) is a versatile chemical used in multiple manufacturing processes. To date, some engineered and non-engineered microbes, such as Escherichia coli, Lactobacillus buchneri, and Clostridium thermosaccharolyticum, have been used to produce 1,2-PDO. In this study, we demonstrated the production of R- and S-1,2-PDO using engineered Lactococcus lactis. The L- and D-lactic acid-producing L. lactis strains NZ9000 and AH1 were transformed with the plasmid pNZ8048-ppy harboring pct, pduP, and yahK genes for 1,2-PDO biosynthesis, resulting in L. lactis LL1 and LL2, respectively. These engineered L. lactis produced S- and R-1,2-PDO at concentrations of 0.69 and 0.50 g/L with 94.4 and 78.0% ee optical purities, respectively, from 1% glucose after 72 h of cultivation. Both 1% mannitol and 1% gluconate were added instead of glucose to the culture of L. lactis LL1 to supply NADH and NADPH to the 1,2-PDO production pathway, resulting in 75% enhancement of S-1,2-PDO production. Production of S-1,2-PDO from 5% mannitol and 5% gluconate was demonstrated using L. lactis LL1 with a pH–stat approach. This resulted in S-1,2-PDO production at a concentration of 1.88 g/L after 96 h of cultivation. To our knowledge, this is the first report on the production of R- and S-1,2-PDO using engineered lactic acid bacteria.

Acid-base Management in Cardiopulmonary Bypass

Cardiopulmonary bypass (CPB) merupakan alat penunjang fungsi sirkulasi dan pernapasan pasien yang biasa digunakan ketika menjalani pembedahan jantung atau pembuluh darah besar. Selama prosedur CPB, kondisi hipotermia dipertahankan untuk menurunkan kebutuhan oksigen dan laju metabolisme. Kondisi hipotermia akan mempengaruhi keseimbangan asam-basa pada tubuh. Manajemen asam-basa selama prosedur CPB dicapai dengan menggunakan metode a-stat atau pH-stat. Pada metode a-stat, manajemen asam-basa dilakukan dengan menjaga pHa 7.4 dan PaCO2 40 mmHg pada suhu 37oC tanpa penambahan CO2 oksigen untuk menjaga total CO2 tetap konstan. Sedangkan, pada metode pH-stat, diberikan CO2 oksigen untuk menjaga PaCO2 40 mmHg dan pHa 7.4 secara in vivo. Masih banyak perdebatan terkait waktu penerapan masing-masing metode. Pada level mikrosirkulasi, manajemen a-stat terbukti memberikan keuntungan pada otak dan mengurangi insidensi postoperative cerebral dysfunction. Sedangkan, metode pH-stat dilaporkan meningkatkan risiko emboli otak, sehingga tidak disarankan untuk pasien yang memiliki risiko tinggi gangguan aliran darah otak. Namun, terdapat pula laporan yang menyatakan pH-stat bermanfaat pada operasi bedah jantung anak. Berdasarkan hal itu, usia pasien dapat menentukan waktu penggunaan metode a-stat dan pH-stat. Satu indikasi primer penggunaan pH-stat adalah selama proses pendinginan saat deep hypothermic circulatory arrest (DHCA), sedangkan metode a-stat lebih baik digunakan selama selective cerebral perfusion (SCP) dan rewarming.

Meat Waste Valorization through Protein Hydrolysis using Different Types of Proteases

Five different commercial proteases (Alcalase 2.4L, Flavourzyme 1000L, Neutrase, Protamex, and PTN) were evaluated for the simultaneous recovery of protein and lipids through hydrolysis. The hydrolysis reaction was monitored using the pH-stat procedure, in which samples were collected after 240 min of hydrolysis using each enzyme. The samples were analyzed for the degree of hydrolysis, protein hydrolysate, collagen, lipids, and fatty acids. A clear relationship was observed between the degree of hydrolysis and the amounts of recovered products. Serine endopeptidases from the microbial source (Alcalase) resulted in the maximum degree of hydrolysis (27.5%), lipid recovery (82.6%), and protein hydrolysates quality (average molecular weight of the hydrolysates = 472 Dalton), followed by formulations of serine protease and metalloprotease from the microbial source (Protamex). Metalloproteases from the microbial source (Neutrase) resulted in maximum collagen recovery (87.1%). Serine endopeptidases from the animal source (PTN) and endo/exopeptidases from the fungal source (Flavourzyme) exhibited an intermediate efficacy between Alcalase and Neutrase. In the case of all proteases, the product fatty acid profile matched well with that of the meat waste, which suggests that no chemical changes occurred in the lipids after the hydrolysis with the evaluated proteases. These results suggest that hydrolysis using proteases could serve as an ecofriendly and viable alternative for obtaining additional value from meat waste.

A cardiopulmonary bypass strategy to support a patient with vein of Galen malformation

We present a dissection of the patent ductus arteriosus and pulmonary artery for surgical repair utilising cardiopulmonary bypass in the setting of vein of Galen malformation. Several strategies were employed to attenuate the cerebral shunt including pH-stat, high cardiac index, restrictive venous drainage, continuous ventilation and deep hypothermic circulatory arrest. The patient recovered from surgery with no apparent neurological sequelae.

pH-Stat Titration: A Rapid Assay for Enzymatic Degradability of Bio-Based Polymers

Bio-based polymers have been suggested as one possible opportunity to counteract the progressive accumulation of microplastics in the environments. The gradual substitution of conventional plastics by bio-based polymers bears a variety of novel materials. The application of bioplastics is determined by their stability and bio-degradability, respectively. With the increasing implementation of bio-based plastics, there is also a demand for rapid and non-elaborate methods to determine their bio-degradability. Here, we propose an improved pH Stat titration assay optimized for bio-based polymers under environmental conditions and controlled temperature. Exemplarily, suspensions of poly(lactic acid) (PLA) and poly(butylene succinate) (PBS) microparticles were incubated with proteolytic and lipolytic enzymes. The rate of hydrolysis, as determined by counter-titration with a diluted base (NaOH), was recorded for two hours. PLA was hydrolyzed by proteolytic enzymes but not by lipase. PBS, in contrast, showed higher hydrolysis rates with lipase than with proteases. The thermal profile of PLA hydrolysis by protease showed an exponential increase from 4 to 30 °C with a temperature quotient Q10 of 5.6. The activation energy was 110 kJ·mol−1. pH-Stat titration proved to be a rapid, sensitive, and reliable procedure supplementing established methods of determining the bio-degradability of polymers under environmental conditions.

An alternative solution for α-linolenic acid supplements: in vitro digestive properties of silkworm pupae oil in a pH-stat system

A brief description of the digestion system in vitro for silkworm pupae oil. Triacylglycerols are hydrolyzed by pancreatic lipase. Meanwhile, the release level and a first-order kinetic model were used to investigate lipid digestion properties.