As the product system of biocatalytic reactions, enzymatic hydrolysate has complex components, including target products, unreacted substrates, enzyme molecules, and various impurities. Membrane technology, with its precise molecular sieving capability, has become the core means for efficient treatment of enzymatic hydrolysate. From ultrafiltration (UF) and microfiltration (MF) to nanofiltration (NF) and reverse osmosis (RO), different membrane technologies exhibit unique advantages in the separation, purification, concentration of enzymatic hydrolysate, and enzyme recovery, promoting technological innovation in the field of bioprocessing.
After the enzymatic reaction is completed, the system often contains impurities such as undissolved solid particles and denatured protein flocs. If these substances directly enter the subsequent treatment process, they will cause membrane fouling or affect product purity. Microfiltration (MF) and ultrafiltration (UF) play important pretreatment roles at this stage.
The pore size of microfiltration membranes is typically 0.1–10 μm. Driven by pressure, they can effectively intercept cell debris, solid particles, and large colloidal impurities in enzymatic hydrolysate. For example, in the treatment of plant protein hydrolysate, filtering through a 0.45 μm cellulose ester microfiltration membrane can remove uncompletely broken plant cell residues from the enzymatic hydrolysis process, reducing the turbidity of the solution from 50 NTU to below 5 NTU, creating clean feed conditions for subsequent ultrafiltration separation. Microfiltration is usually operated at room temperature and low pressure (0.1–0.3 MPa), with low energy consumption, and can adopt cross-flow filtration mode to reduce membrane surface fouling and maintain stable flux.
The molecular weight cutoff (MWCO) range of ultrafiltration membranes is 1,000–100,000 Da. In addition to further removing fine colloidal particles that are difficult to intercept by microfiltration, it is more importantly capable of preliminary fractionation of macromolecular components in enzymatic hydrolysate. Taking soybean protein hydrolysate as an example, using a polysulfone ultrafiltration membrane with a MWCO of 30,000 Da can intercept uncompletely hydrolyzed soybean proteins (molecular weight > 50,000 Da) and enzyme molecules (molecular weight about 20,000–50,000 Da), while polypeptides and amino acids with a molecular weight < 30,000 Da pass through the membrane into the filtrate, achieving the separation of enzymatic hydrolysis products from macromolecular impurities. During ultrafiltration, the operating pressure is generally controlled at 0.2–0.6 MPa, the temperature at 25–40°C. By adjusting the flow rate (1–3 m/s) to reduce the concentration polarization phenomenon, the membrane flux can be stabilized at 10–30 L/(m²·h).
After the enzymatic reaction ends, recovering active enzymes and realizing their separation from products are key links to reduce production costs, where ultrafiltration technology shows irreplaceable advantages. Since most industrial enzymes (such as α-amylase, neutral protease) have a molecular weight between 10,000 and 100,000 Da, while the molecular weight of enzymatic hydrolysis products (such as amino acids, oligosaccharides) is usually less than 10,000 Da, selecting an ultrafiltration membrane with a suitable MWCO can achieve effective separation of the two.
In the process of producing glucose by hydrolyzing starch catalyzed by glucoamylase, using a polyacrylonitrile ultrafiltration membrane with a MWCO of 10,000 Da can intercept glucoamylase with a molecular weight of about 60,000 Da and recycle it to the reactor, while the generated glucose (molecular weight 180 Da) passes through the membrane into the product liquid. The enzyme recovery rate can reach more than 95%, and the glucose transmittance exceeds 98%. This "in-situ enzyme recovery" process not only reduces enzyme usage (cost reduction by 30%–50%) but also avoids the influence of enzymes on the subsequent crystallization of products. To further improve the enzyme recovery efficiency, tangential flow ultrafiltration (TFF) technology can be adopted. Through the high-speed flow of the feed liquid on the membrane surface, the adsorption and deposition of enzyme molecules on the membrane surface are reduced, so that the long-term operation flux decay rate of the membrane is controlled within 15%.
When the target products in enzymatic hydrolysate are low-molecular-weight substances (such as nucleotides, small peptides) and inorganic salts or small-molecular impurities need to be removed, nanofiltration (NF) technology becomes an ideal choice. The MWCO of nanofiltration membranes is about 200–1,000 Da, and their surfaces are charged (such as negative charge). In addition to size sieving, they can also separate charged molecules through charge repulsion, a feature that gives them unique advantages in the field of desalination of enzymatic hydrolysate.
In the process of producing 5'-nucleotides by enzymatic hydrolysis of yeast RNA, after removing macromolecular proteins from the enzymatic hydrolysate by ultrafiltration, a negatively charged nanofiltration membrane (such as DK series) is used for desalination. Since 5'-nucleotides (such as 5'-AMP) are negatively charged under neutral conditions, while Na⁺ and Cl⁻ dissociated from inorganic salts (such as NaCl) are positively and negatively charged respectively, the nanofiltration membrane has a 截留率 (retention rate) of more than 90% for Na⁺ and Cl⁻, and a retention rate of less than 5% for 5'-AMP, thus achieving efficient desalination of the product. After treatment, the salt concentration in the enzymatic hydrolysate is reduced from 5,000 mg/L to below 500 mg/L, and the purity of 5'-AMP is increased from 75% to 92%, laying a foundation for subsequent crystallization and purification. Nanofiltration is usually operated at a pressure of 0.5–1.5 MPa and a temperature of 30–50°C, with a water flux of 5–15 L/(m²・h). Moreover, since nanofiltration membranes have a higher retention rate for divalent ions than monovalent ions, selective desalination can be achieved in some cases.
For enzymatic hydrolysis products that need to be recovered at high concentration (such as amino acid solutions, functional oligosaccharides), reverse osmosis (RO) technology can achieve a low-energy consumption concentration process. The MWCO of reverse osmosis membranes is less than 200 Da, which can intercept almost all organic molecules and inorganic salts. Driven by high pressure (1–10 MPa), only water molecules are allowed to pass through, so as to achieve the purpose of high concentration.
In the treatment of enzymatic hydrolysis products of glutamic acid fermentation broth, the energy consumption of the traditional evaporation concentration method is as high as 800–1,000 kWh/ton of water, while using aromatic polyamide reverse osmosis membranes for concentration at a pressure of 1.5–2.0 MPa can concentrate the glutamic acid solution from 5% to more than 20%, with energy consumption only 30%–40% of that of the evaporation method. At the same time, the reverse osmosis process is carried out at room temperature, avoiding the damage of high temperature to heat-sensitive products (such as certain active peptides). To solve the problem of membrane fouling during high-concentration concentration, a multi-stage reverse osmosis system can be adopted. The front-stage membrane processes the low-concentration feed liquid, and the rear-stage membrane processes the concentrated liquid, which not only ensures the flux but also improves the concentration multiple. At present, the application of reverse osmosis in the concentration of enzymatic hydrolysate has been expanded from a single solute system to a complex mixed system. Through the modification of membrane materials (such as hydrophilic coatings) and the optimization of operating conditions, the retention rate of organic solutes can be maintained above 99%, and the water flux is stable at 1–5 L/(m²·h).
Single membrane technology often 难以 (is difficult to) meet all the needs of enzymatic hydrolysate treatment. Integrating different membrane technologies with traditional processes can construct a more efficient and environmentally friendly production process. A typical membrane integration process such as the "microfiltration - ultrafiltration - nanofiltration - reverse osmosis" combination has shown significant advantages in the production of functional polypeptides.
Taking the preparation of antioxidant peptides by enzymatic hydrolysis of fish protein as an example, first, microfiltration is used to remove fat particles and insoluble impurities in the fish protein hydrolysate, and then an ultrafiltration membrane with a MWCO of 5,000 Da is used to separate small peptides with antioxidant activity (molecular weight < 5,000 Da), while intercepting and recovering proteases; the ultrafiltration permeate is desalted by a nanofiltration membrane to remove salts and small-molecular impurities, and then concentrated by reverse osmosis to obtain a high-concentration antioxidant peptide solution. Finally, peptide powder with a purity of more than 90% is obtained by spray drying. Compared with the traditional "centrifugation - precipitation - evaporation" process, the entire process increases the product recovery rate by 20%–30%, reduces energy consumption by 40%, and reduces wastewater discharge by 60%. In addition, the membrane integration process can also be coupled with the enzymatic reaction process to form a "reaction-separation" integrated system. For example, placing the ultrafiltration membrane module directly in the enzymatic reactor to separate products in real time and recover enzymes can increase the reaction conversion rate from 70%–80% of the traditional batch process to more than 95%, significantly shortening the production cycle.
Although membrane technology has shown strong advantages in enzymatic hydrolysate treatment, it still faces many challenges in practical applications. Membrane fouling is the most prominent problem. Macromolecular substances such as proteins and polysaccharides in enzymatic hydrolysate are prone to form a gel layer or adsorb and deposit on the membrane surface, leading to a decrease in flux and separation efficiency. According to statistics, when ultrafiltration is directly performed on the unpretreated enzymatic hydrolysate, the membrane flux may decay by more than 50% within 1 hour. In addition, for enzymatic hydrolysis systems with complex components (such as containing multiple products with similar molecular weights), the separation selectivity of a single membrane technology is limited, making it difficult to achieve precise separation; membrane technologies with high-pressure operation (such as reverse osmosis) still have high energy consumption, limiting their application in low-value-added enzymatic hydrolysis products.
In response to these challenges, the future development of membrane technology in enzymatic hydrolysate treatment will show the following trends: first, developing anti-fouling membrane materials, reducing the adsorption of macromolecular substances by grafting hydrophilic groups (such as polyethylene glycol), introducing antibacterial coatings, or constructing rough surface structures; second, designing intelligent responsive membranes, such as pH-responsive and temperature-responsive membranes, regulating membrane pore size or charge through changes in external conditions to achieve dynamic optimized separation; third, deepening the development of membrane integration processes, combining membrane technology with separation methods such as affinity chromatography and electrophoresis to construct a multi-dimensional separation system and improve the separation accuracy of complex enzymatic hydrolysate; fourth, exploring new membrane technologies, such as membrane contactors and membrane distillation, to expand the application boundaries of membrane technology in enzymatic hydrolysate treatment.
From laboratory research to industrial production, membrane technology is profoundly changing the mode of enzymatic hydrolysate treatment. Its precise molecular sieving ability, high separation efficiency, and green environmental protection characteristics make it a key link connecting enzymatic reactions and final products. With the continuous innovation of membrane materials and processes, the application of membrane technology in enzymatic hydrolysate treatment will surely be more extensive, providing strong support for the high-quality development of bio-pharmaceuticals, food processing, fine chemicals, and other fields.
