Optimal Hydrolysis Conditions Research Articles

Preparation, identification and screening of anti-osteoporosis milk-derived peptides: Intervention effects in osteoporosis rats

To identify milk-derived peptides with both antioxidant and calcium absorption activities in combating osteoporosis, we employed a comprehensive screening approach that included virtual enzymatic hydrolysis, molecular docking, and cellular experiments using osteoblasts. Under the optimal conditions for dual-enzyme hydrolysis, the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical scavenging rate and soluble calcium binding capacity of the milk-derived peptides were 19.69% and 0.6965 μg/mL, respectively. Six peptide segments, namely KEDVPSER, HKEMPFPK, YPSYG, EDVPSE, VPQLE, and IPAVF, were identified through UPLC-Q-Exactive Orbitrap MS and molecular docking for further validation. Among the peptides, YPSYG significantly promoted the proliferation of MC3T3-E1 cells both with and without CaCl2 (P < 0.05), increasing proliferation by 38.27% and 20.67%, respectively, compared to the control group. Additionally, YPSYG significantly improved proliferation after H2O2-induced oxidative damage (P < 0.05), with a 38.23% higher rate than the model group. Compared with rats in the osteoporosis model group, YPSYG significantly enhanced serum alkaline phosphatase (ALP) and N-terminal propeptide of type I procollagen in rats (s-PINP) levels and decreased tartrate-resistant acid phosphatase (TRAP) levels (P < 0.05). Furthermore, milk-derived peptides and YPSYG significantly increased the bone weight index, maximum load, and bending energy of the femur and tibia in osteoporotic rats (P < 0.05). Additionally, these peptides significantly reduced the number of osteoclasts in the metaphysis of the femur and tibia in osteoporotic rats and alleviated microstructural damage. This study confirmed that milk-derived peptides, including YPSYG, effectively promoted bone formation and improved bone microstructure in osteoporotic rats. These findings provided a foundation for developing functional foods for elderly bone health.

Enzymatic Hydrolysis as an Effective Method for Obtaining Wheat Gluten Hydrolysates Combining Beneficial Functional Properties with Health-Promoting Potential

The byproduct from wheat starch production contains approximately 70% gluten (WG) and is an inexpensive but demanding protein raw material for the food industry. This study attempted to determine the optimal hydrolysis conditions for such raw material to obtain peptides combining beneficial functional characteristics with health-promoting activity. The proteases Bromelain, Alcalase, Flavourzyme, and a protease from A. saitoi were used for hydrolysis. It was shown that the tested proteases differ both in terms of the effective hydrolysis conditions of gluten and the profile of the released hydrolysates. Bromelain was particularly effective in converting gluten into peptides, combining beneficial health and functional properties. It achieved maximum activity (189 U/g) against WG at pH 6 and 60 °C, and the best-balanced peptides in terms of desired properties were released at a dose of 2.5 U/g. These peptides were free from most allergenic epitopes, effectively inhibited ACE, and, at 0.34 g, were equivalent to the approved dose of BHT. Their emulsifying activity was higher than that of gluten, and the foaming formation and stabilization potential exceeded that of ovalbumin by 10% and 19%, respectively. It seems that Bromelain-released WG hydrolysates are a promising candidate for a safe fat stabilizer and egg white substitute.

Improved Sugar Recovery from Mandarin Peel under Optimal Enzymatic Hydrolysis Conditions and Application to Bioethanol Production

Improved Sugar Recovery from Mandarin Peel under Optimal Enzymatic Hydrolysis Conditions and Application to Bioethanol Production

Optimization of the Preparation Process and Ameliorative Efficacy in Osteoporotic Rats of Peptide-Calcium Chelates from Skipjack Tuna (Katsuwonus pelamis) Meat.

This study aimed to establish the preparation process of peptide-calcium chelates (TMP-Ca) using skipjack tuna meat and investigate the function and mechanism of TMP-Ca in an osteoporosis model of rats. The results indicated that trypsin is more suitable for preparing the Ca-chelating hydrolysates of tuna meat, and the optimal hydrolysis conditions were derived as follows: digestion time 4 h, material-liquid ratio 1:10, and enzyme dose 3%. The conditions for chelating Ca with tuna meat hydrolysate were optimized to be chelation time 50 min, temperature 50 °C, pH 8.0, and a peptide-Ca ratio 1:10. The prepared hydrolysate was subjected to ultrafiltration, and the fraction (TMP) (MW <1 kDa) showed the highest Ca chelation rate (51.27 ± 1.42%) and was made into the peptide-Ca chelates (TMP-Ca). In osteoporotic rats, TMP-Ca significantly improved the decrease in ovarian indexes caused by retinoic acid. It also elevated serum Ca, phosphorus, and bone turnover indexes, increased the number of bone trabeculae, and improved bone microstructure. In addition, we confirmed that TMP-Ca could regulate the OPG/TRAF6 pathway to reduce osteoclast differentiation, inhibit bone resorption, and promote bone formation. Therefore, TMP-Ca could significantly ameliorate osteoporosis, and this study provides a functional component for the preparation of healthcare products using skipjack tuna meat to treat osteoporosis.

Effects and optimization of hydrolysis conditions on the diameter distance ratio for metatitanic acid

Controlling and predicting the narrow particle size distribution of metatitanic acid could help to improve pigment and application performances of TiO2. To prepare metatitanic acid with narrow particle size distribution, the hydrolysis conditions of industrial TiOSO4 solution were optimized by using the response surface methodology of Box-Behnken design method. The effects of hydrolysis conditions such as TiO2 concentration, aging time and hydrolysis time on the diameter distance ratio and structure of metatitanic acid were investigated. The prediction model including the hydrolysis variables with good fitting result was established to predict the diameter distance ratio or optimize the hydrolysis variable values. The regression equation model was significant and reliable with actual correlation coefficient R2 of 0.9989. The hydrolysis conditions greatly influenced the quality and quantity of hydrolysis induced crystal nucleus, hydrolysis rate, completeness degree of hydrolysis, crystallization growth and aggregation rate, and eventually affected the particle size distribution of metatitanic acid. The synergistic interaction for TiO2 concentration and hydrolysis time was very significant. The verification experiments confirmed that the diameter distance ratio could reach the average value of 1.100 under the optimal conditions. The prepared metatitanic acid had anatase TiO2 phase structure, with average particle size of 1.621–1.819 µm. And this helped to improve the high quality and green development of the titanium dioxide industry.

Valorization of Yellowfin Tuna Tails: From Proteolytic Enzyme Production to Gelatin and Antioxidant Hydrolysate Extraction.

This study investigates the valorization potential of yellowfin tuna (Thunnus albacares) tails to produce high-value commercial products. Firstly, the tuna tails were placed in a perforated stainless-steel cylinder, and hydraulic pressure was applied to separate the skin from the muscle in the tails. The extracted muscle was then utilized as a nitrogen source for the growth of the proteolytic enzyme producer Bacillus subtilis, while the skins were employed for gelatin extraction. The proteases from B. subtilis were partially purified and used to produce antioxidant peptides from the obtained gelatin. The gelatin formed a gel upon cooling, with gelling and melting temperatures of 16 °C and 22 °C, respectively, and a Bloom strength of approximately 160. Response Surface Methodology (RSM) was employed to determine the optimal hydrolysis conditions to achieve the highest antioxidant activity (35.96% measured as DPPH radical scavenging activity), which were 50 °C and 6.5 IU of enzyme. The findings emphasize the importance of an integrated approach to maximize the value of tuna by-products, promoting sustainability within the framework of a circular bioeconomy. Overall, these results contribute to the efficient utilization of tuna by-products, waste reduction, and enhanced economic viability of the tuna industry.

Optimizing the Conditions of Pretreatment and Enzymatic Hydrolysis of Sugarcane Bagasse for Bioethanol Production.

The agricultural waste sugarcane bagasse (SCB) is a kind of plentiful biomass resource. In this study, different pretreatment methods (NaOH, H2SO4, and sodium percarbonate/glycerol) were utilized and compared. Among the three pretreatment methods, NaOH pretreatment was the most optimal method. Response surface methodology (RSM) was utilized to optimize NaOH pretreatment conditions. After optimization by RSM, the solid yield and lignin removal were 54.60 and 82.30% under the treatment of 1% NaOH, a time of 60 min, and a solid-to-liquid ratio of 1:15, respectively. Then, the enzymolysis conditions of cellulase for NaOH-treated SCB were optimized by RSM. Under the optimal enzymatic hydrolysis conditions (an enzyme dose of 18 FPU/g, a time of 64 h, and a solid-to-liquid ratio of 1:30), the actual yield of reducing sugar in the enzyme-treated hydrolysate was 443.52 mg/g SCB with a cellulose conversion rate of 85.33%. A bacterium, namely, Bacillus sp. EtOH, which produced ethanol and Baijiu aroma substances, was isolated from the high-temperature Daqu of Danquan Baijiu in our previous study. At last, when the strain EtOH was cultured for 36 h in a fermentation medium (reducing sugar from cellulase-treated SCB hydrolysate, yeast extract, and peptone), ethanol concentration reached 2.769 g/L (0.353%, v/v). The sugar-to-ethanol and SCB-to-ethanol yields were 13.85 and 11.81% in this study, respectively. In brief, after NaOH pretreatment, 1 g of original SCB produced 0.5460 g of NaOH-treated SCB. Then, after the enzymatic hydrolysis, reducing sugar yield (443.52 mg/g SCB) was obtained. Our study provided a suitable method for bioethanol production from SCB, which achieved efficient resource utilization of agricultural waste SCB.

Non-ionic surfactant PEG: Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate

To enhance the enzymatic digestibility of polyethylene terephthalate (PET), which is highly oriented and crystallized, a polyethylene glycol (PEG) surfactant of varying molecular weights was utilized to improve the stability of mutant cutinase from Humicola insolens (HiC) and to increase the accessibility of the enzyme to the substrate. Leveraging the optimal conditions for HiC hydrolysis of PET, the introduction of 1 % w/v PEG significantly increased the yield of PET hydrolysis products. PEG600 was particularly effective, increasing the yield by 64.58 % compared to using HiC alone. Moreover, the mechanisms by which PEG600 and PEG6000 enhance enzyme digestion were extensively examined using circular dichroism and fluorescence spectroscopy. The results from CD and fluorescence analyses indicated that PEG alters the protein conformation, thereby affecting the catalytic effect of the enzyme. Moreover, PEG improved the affinity between HiC and PET by lowering the surface tension of the solution, substantially enhancing PET hydrolysis. This study suggests that PEG holds considerable promise as an enzyme protector, significantly aiding in the hydrophilic modification and degradation of PET in an environmentally friendly and sustainable manner.

Response surface optimization in determining hydrolysis reaction on Barramundi scales gelatin using the box behnken design

Gelatin hydrolysate is a product with a wide range of benefits in pharmaceutical and food applications. Therefore, this study analyzes the best hydrolysis condition to make Barramundi (Lates calcarifer) scales gelatin hydrolysate using ultrasonication and lime juice. The conditions with the best degree of hydrolysis (DH) were optimized using the Response Surface Method (RSM) with Box Behnken Design (BBD). The effects of 3 independent variables, namely time, temperature, and enzyme concentrations, were then examined for model optimization. Based on the extraction method, DH varied between 57.40% and 68.60%. The equation characterizing the variables and DH correlation was DH = 0.01324 – 0.0012A + 0.0013B – 0.0028C -0.0004AB + 0.0019AC – 0.001BC – 0.0004A2 – 0,0042B2 – 0.0012C2 , A, B, and C were time, temperature, and E/S concentration. In addition, the optimized hydrolysis was performed at 31.03 mins, 56.84оC temperature, 1.5% E/S concentration, and pH 8.0. At these optimal conditions, alkaline protease hydrolysis yielded a 1.000 desirability value, with a 64.83% DH. Amino acid (AA) with the highest value in Barramundi scales gelatin hydrolysate was lysine (53.03 g/100 g), while isoleucine (0.05 g/100 g) had the lowest value. Based on the results, optimal hydrolysis conditions could be an excellent reference to increase the potential of Barramundi scales gelatin hydrolysate in developing future functional foods with high amino acids, particularly lysine.

Optimization of Sorghum Starch (Sorghum bicolor L.) Partial Hydrolysis using Microwave-Assisted Acetic Acid Catalyst

Hydrolysis process is relatively slow, requiring the use of catalyst and microwave assistance to accelerate the reaction. There fore, this study aimed to determine the optimum conditions for partial hydrolysis of sorghum starch (Sorghum bicolor L.) using acetic acid as a microwave-assisted catalyst to produce maltodextrin. The experiment was carried out in several stages, namely gelatinization, liquefaction, and drying, while Response Surface Method (RSM) was used for variable design and data analysis. Hydrolysis process was carried out with three independent variables, including acetic acid concentration (9%, 12%, and 15%), microwave power (300 watts, 400 watts, and 500 watts), and liquefaction time (35 minutes, 45 minutes, and 55 minutes). The results showed that the highest DE (Dextrose Equivalent) value of maltodextrin was found at 17.04% acid concentration, 400 watts microwave power, and 45 minutes liquefaction time, valued at 18.921 ± 0.099. The optimum dextrose equivalent of 17.763 would be achieved at 16.879% acetic acid concentration, 390.233 watts of microwave power, and 47.055 minutes of liquefaction time. This study introduces the innovation of using acetic acid as a catalyst and microwave assistance during the gelatinization stage.

Turning discarded blue shark (Prionace glauca) skin into a valuable nutraceutical resource: An enzymatic collagen hydrolysate

Marine-derived collagen, particularly from blue shark (Prionace glauca) skin, represents a sustainable resource for the nutraceutical industry, yet its effective utilization remains underexplored. This study aims to optimise the enzymatic hydrolysis of blue shark skin collagen using alcalase and bromelain to enhance the bioactive properties of the resultant hydrolysates. We employed a multifactorial experimental design to determine the optimal hydrolysis conditions, assessing factors including enzyme concentration, pH, and temperature. The alcalase-treated hydrolysates demonstrated superior antioxidant and anti-inflammatory activities compared to those treated with bromelain, with increased solubilised proteins and a higher degree of hydrolysis. Notably, peptide profiles indicated that alcalase hydrolysates favoured the production of smaller peptides, suggesting enhanced bioavailability and digestive stability. In vitro gastrointestinal simulations demonstrated the functional stability of these peptides, indicating that while they undergo structural changes during digestion, their potential for gastrointestinal health remains significant. Our findings highlight the feasibility of converting blue shark skin, a commonly discarded by-product, into valuable nutraceutical ingredients, thus contributing to marine sustainability and waste reduction This research advances the biotechnological application of marine collagen and opens avenues for developing functional foods and pharmaceuticals.

Preparation of xylooligosaccharides from rice husks and their structural characterization, antioxidant activity, and probiotic properties

Rice husks are rich in xylan, which can be hydrolyzed by xylanase to form xylooligosaccharides (XOS). XOS are a functional oligosaccharide such as improving gut microbiota and antioxidant properties. In this study, the structure and functional characteristics of XOS were studied. The optimal xylanase hydrolysis conditions through response surface methodology (RSM) were: xylanase dosage of 3000 U/g, hydrolysis time of 3 h, hydrolysis temperature of 50 °C. Under this condition, the yield of XOS was 150.9 mg/g. The TG-DTG curve showed that XOS began to decompose at around 200 °C. When the concentration of XOS reached 1.0 g/L, the clearance rate of DPPH reached 65.76 %, and the scavenging rate of OH reached 62.10 %, while the clearance rate of ABTS free radicals reached 97.70 %, which was equivalent to the clearance rate of VC. XOS had a proliferative effect on four probiotics: Lactobacillus plantarum, Lactobacillus brucelli, Lactobacillus acidophilus, and Lactobacillus rhamnosus. However, the further experiments are needed to explore the improvement effect of XOS on human gut microbiota, laying a foundation for the effective utilization of XOS. XOS have a wide range of sources, low price, and broad development prospects. The reasonable utilization of XOS can bring greater economic benefits.

Recovery of Ellagic Acid from Pomegranate Peels with the Aid of Ultrasound-Assisted Alkaline Hydrolysis.

The pomegranate processing industry generates worldwide enormous amounts of by-products, such as pomegranate peels (PPs), which constitute a rich source of phenolic compounds. In this view, PPs could be exploited as a sustainable source of ellagic acid, which is a compound that possesses various biological actions. The present study aimed at the liberation of ellagic acid from its bound forms via ultrasound-assisted alkaline hydrolysis, which was optimized using response surface methodology. The effects of duration of sonication, solvent:solid ratio, and NaOH concentration on total phenol content (TPC), antioxidant activity, and punicalagin and ellagic acid content were investigated. Using the optimum hydrolysis conditions (i.e., 32 min, 1:48 v/w, 1.5 mol/L NaOH), the experimental responses were found to be TCP: 4230 ± 190 mg GAE/100 g dry PPs; AABTS: 32,398 ± 1817 µmol Trolox/100 g dry PPs; ACUPRAC: 29,816 ± 1955 µmol Trolox/100 g dry PPs; 59 ± 3 mg punicalagin/100 g dry PPs; and 1457 ± 71 mg ellagic acid/100 g dry PPs. LC-QTOF-MS and GC-MS analysis of the obtained PP extract revealed the presence of various phenolic compounds (e.g., ellagic acid), organic acids (e.g., citric acid), sugars (e.g., fructose) and amino acids (e.g., glycine). The proposed methodology could be of use for food, pharmaceutical, and cosmetics applications, thus reinforcing local economies.

Integrated biorefinery approach for utilization of wood waste into levulinic acid and 2-Phenylethanol production under mild treatment conditions

In a bid to explore the on-site biorefinery approach for conversion of forestry residues, lignocellulosic biomass into value-added products was studied. The bark white pine wood was subjected to the microwave technique of fast and slow hydrolysis under varying acid and biomass concentrations to produce levulinic acid (LA). The HCl (2% v/v) and plant biomass (1% w/v) were identified as the optimum conditions for fast wood hydrolysis (270 ºC for 12 sec), which led to maximum LA yield of 446.68 g/kgPB. The proposed sustainable approach is mild, quick, and utilized a very low concentration of the HCl for the production of LA. The hydrolysate was used as a medium for Kluyveromyces marxianus growth to produce 2-phenylethanol (2-PE). K. marxianus used 74–95% of furfural from hydrolysate as a co-substrate to grow. The proposed model of the integrated biorefinery is an affordable on-site approach of using forest waste into localized solutions to produce LA and 2-PE.

Immobilization of Cutinase from Fussarium oxysporum into Sea Sand Matrix for Catalytic Degradation of Polyethylene Terephthalate (PET)

Cutinase is an enzyme that can be used as a catalyst in the hydrolysis reaction of polyethylene terephthalate (PET) waste into terephthalic acid and ethylene glycol. Generally, cutinase has low stability in its free form. Therefore, to increase cutinase stability, cutinase needs to be immobilized. In this study, cutinase was immobilized into a sea sand matrix by adsorption technique. The purpose of this study was to determine the optimum conditions of PET hydrolysis (pH, temperature, and incubation time), and to study the ability of immobilized cutinase for catalytic degradation of PET. The results show that immobilized cutinase has optimum conditions at pH 8, temperature of 50 °C, and incubation time of 24 hrs. Moreover, the immobilized cutinase can be used for up to three cycles with a residual activity percentage of 66.2% and an activity value of 23.224 nmol. g-1. min-1. The total enzyme activity of immobilized cutinase after being used in three cycles was 89.99 nmol. g-1. min-1 or 28.6% of its free enzyme activity.

Optimization of hydrolysis conditions of alginate based on high performance liquid chromatography

Alginate is the most abundant polysaccharide compound in brown algae, which is widely used in various fields. At present, the determination of the content of alginate is mostly carried out using sulfuric acid and trifluoroacetic acid hydrolysis followed by the determination of the content, but the results are not satisfactory, and there are problems such as low hydrolysis degree and low recovery rate. Therefore, in this study, based on the optimization of high performance liquid chromatographic conditions for pre-column derivatization of 1-phenyl-3-methyl-5-pyrazolone (PMP), the hydrolysis effects of sulfuric acid, trifluoroacetic acid (TFA), oxalic acid, and formic acid were compared and the hydrolysis conditions were optimized. The results showed that formic acid was the best hydrolyzing acid. The optimal hydrolysis conditions were 95 % formic acid at 110 °C for 10 h. The hydrolysis effect was stable, with high recovery and low destruction of monosaccharides, which made it possible to introduce formic acid into the subsequent polysaccharide hydrolysis. The pre-column derivatization high performance liquid chromatography method established in this study was accurate and reliable, and the hydrolysis acid with better effect was screened, which provided a theoretical basis for the subsequent determination of alginate content.

Exploration and Improvement of Acid Hydrolysis Conditions for Inulin-Type Fructans Monosaccharide Composition Analysis: Monosaccharide Recovery and By-Product Identification.

Acid hydrolysis serves as the primary method for determining the monosaccharide composition of polysaccharides. However, inappropriate acid hydrolysis conditions may catalyze the breakdown of monosaccharides such as fructans (Fru), generating non-sugar by-products that affect the accuracy of monosaccharide composition analysis. In this study, we determined the monosaccharide recovery rate and non-sugar by-product formation of inulin-type fructan (ITF) and Fru under varied acid hydrolysis conditions using HPAEC-PAD and UPLC-Triple-TOF/MS, respectively. The results revealed significant variations in the recovery rate of Fru within ITF under different hydrolysis conditions, while glucose remained relatively stable. Optimal hydrolysis conditions for achieving a relatively high monosaccharide recovery rate for ITF entailed 80 °C, 2 h, and 1 M sulfuric acid. Furthermore, we validated the stability of Fru during acid hydrolysis. The results indicated that Fru experienced significant degradation with an increasing temperature and acid concentration, with a pronounced decrease observed when the temperature exceeds 100 °C or the H2SO4 concentration surpasses 2 M. Finally, three common by-products associated with Fru degradation, namely 5-hydroxymethyl-2-furaldehyde, 5-methyl-2-furaldehyde, and furfural, were identified in both Fru and ITF hydrolysis processes. These findings revealed that the degradation of Fru under acidic conditions was a vital factor leading to inaccuracies in determining the Fru content during ITF monosaccharide analysis.

Alcalase-Based Chickpea (Cicer arietinum L.) Protein Hydrolysates Efficiently Reduce Systolic Blood Pressure in Spontaneously Hypertensive Rats.

Studies on antihypertensive chickpea protein hydrolysates have rarely performed in vivo evaluations, limiting the entry of such hydrolysates into functional food development and clinical trials. Thus, our aim was to optimize the hydrolysis conditions to produce an alcalase-based chickpea hydrolysate with a hypotensive effect in vivo at convenient oral doses. The hydrolysis reaction time, temperature, and alcalase/substrate concentration were optimized using a response surface analysis (RSA). ACE-I inhibition was the response variable. The optimized hydrolysis conditions were time = 0.5 h, temperature = 40 °C, and E/S concentration = 0.254 (U/g). The IC50 of the optimized hydrolysate (OCPH) was 0.358 mg/mL. Five hydrolysates from the RSA worksheet (one of them obtained after 5 min of hydrolysis (CPH15)) had an ACE-I inhibitory potential similar to that of OCPH (p > 0.05). At 50 mg/kg doses, OCPH and CPH15 promoted a clinically relevant hypotensive effect in spontaneously hypertensive rats, up to -47.35 mmHg and -28.95 mmHg, respectively (p < 0.05 vs. negative control). Furthermore, the hypotensive effect was sustained for at least 7 h post-supplementation. Overall, OCPH and CPH15 are promising ingredients for functional food development and as test materials for clinical trials.

Determination of Optimum Conditions of pH and Temperature on the Saccharification of Cassava Starch by Amyloglucosidase

Saccharification is the process of breaking complex carbohydrate into its monosaccharide components. The present study however was carried out to determine the optimum conditions of pH and temperature on the saccharification of cassava starch by amyloglucosidase. The optimum conditions of cassava starch hydrolysis was determined by using a pure culture of a thermostable amyloglucosidase for saccharification, and the activities of the enzyme determined at varying pH, temperature and time. Results showed that sample dry weight significantly decreased with respect to increased value of pH, temperature and time, while reducing sugar and dextrose equivalent significantly increased with respect to increased time. The optimal reducing sugar and dextrose equivalent were 74.23% and 96.41 DE, respectively at pH 4.5, 55 °C and 72 h. The glucose obtained from this process may serve as a substrate in fructose syrup production.

Structural Characterization and Immunological Activity of Polysaccharide Degradation Products from Phlebopus portentosus

Phlebopus portentosus is an edible and medicinal mushroom with a delicious taste and high nutritional value. The oligosaccharides derived from P. portentosus may be the material basis for its biological activity. The degradation of polysaccharide and the maintenance of its activity after degradation are key steps in related research. This study applied an acid degradation method to prepare P. portentosus refined polysaccharide (PPRP) with a smaller molecular weight, and the optimal hydrolysis conditions determined were a temperature of 80 °C, an acid concentration of 2 mol/L, and a hydrolysis time of 2 h. The polysaccharide structure and immune activity were then further investigated. The results showed that the PPRP comprised two fractions with approximate weights of 61,600 Da and 5500 Da. The monosaccharide composition of PPRP was mannose, rhamnose, glucose, and galactose, with a molar ratio of 1.00: 22.24: 2.93: 1.03. The major functional groups included O-H, C-H, C-O, and C-O-C. The glycosidic bond types were mainly α- and β-glycosidic bonds. Cell experiments demonstrated that PPRP could significantly increase the proliferation of macrophages and enhance the cytotoxicity of NK cells. Moreover, PPRP also significantly promoted the proliferation of B lymphocytes and T lymphocytes, especially at a concentration of 200 μg/mL. This study furnishes scientific evidence underlining the significant potential of PPRP in immune activity, thereby serving as a material basis and scientific bedrock for further investigations into the mechanism of P. portentosus oligosaccharide activity.