Extraction Of Polysaccharides Research Articles

Extraction and characterization of polysaccharides from Schisandra sphenanthera fruit by Lactobacillus plantarum CICC 23121-assisted fermentation

Improving the yield of polysaccharides extracted from Schisandra sphenanthera is a major challenge in traditional Chinese medicinal plants. In this study, we investigated the potential of Lactobacillus plantarum CICC 23121-assisted fermentation as an extraction tool for S. sphenanthera polysaccharides (SSP). We observed that 11.12 ± 0.28 % of polysaccharides were extracted from S. sphenanthera using strain CICC 23121 -assisted fermentation (F-SSP), which was 53.38 % higher than that using hot water extraction (NF-SSP). The optimized parameters were a fermentation time of 15.5 h, substrate concentration of 4 %, and inoculum size of 3 %. Lactic acid produced by strain CICC 23121 increased the release of intracellular polysaccharides by breaking down cell walls. Compared to NF-SSP, F-SSP contained higher and lower total carbohydrate and protein contents, respectively, and its monosaccharide composition was the same as that of NF-SSP; however, their distributions were different. F-SSP had a higher molecular weight, better aqueous stability, and looser surface morphology, and strain CICC 23121-assisted fermentation did not change the molecular structure of SSP. Both NF-SSP and F-SSP showed the potential to regulate human intestinal microflora. Our findings revealed that strain CICC 23121-assisted fermentation is an efficient method for extracting S. sphenanthera polysaccharides without affecting their physicochemical and bioactive properties.

Extraction of Polysaccharides from Shiitake Mushroom Based on Pineapple Enzymatic Technology

The pineapple enzymatic technique was used to extract polysaccharides from shiitake mushrooms, with glucose serving as the standard for calculating the yield of the polysaccharides. The effects of acidity, alkalinity, extraction time, extraction temperature, enzyme dosage, and number of extractions on the extraction rate of mushroom polysaccharides were investigated under the control of a single factor. Orthogonal tests showed that the optimal process combination for the extraction of mushroom polysaccharides under the influence of multiple factors was pH 5, an extraction time of 1.5 hours, extraction temperature of 45°C, enzyme dosage of 0.8/100 g, and the number of extraction times of 3. The yield of mushroom polysaccharides under optimal processing conditions could reach 6.47%. The method is easy to operate, accurate, highly efficient, and it is a non-polluting and cost-effective process for extracting shiitake polysaccharides.

Polysaccharide extract of Spirulina sp. increases effector immune-cell killing activities against cholangiocarcinoma.

Cyanobacteria and algae serving as promising food supplements have recently garnered attention for their emerging potential in anti-cancer activity. Cholangiocarcinoma (CCA) or bile duct cancer is one of the top-leading cancers affecting people, particularly in Asian continent. With patients exhibiting no or minimal symptoms in the early stages, advanced CCA is often diagnosed, and primary treatments such as surgery may not be suitable. Discovery of natural bioactive compounds for cancer treatments have, thus, attracted attention as one of the effective means to combat CCA or to supplement primary treatments. In this work, ethanolic and polysaccharide extracts of cyanobacteria and algae were tested for their cytotoxicity against 2 CCA cell lines (KKU055 and KKU213A). The ethanolic extracts from Leptolyngbya sp. and Chlorella sp. demonstrated growth inhibition of both CCA cell lines, with IC50 values of 0.658 mg/mL and 0.687 mg/mL for KKU055, and 0.656 mg/mL and 0.450 mg/mL for KKU213A. In contrast, only the polysaccharide extracts from Sargassum spp. exhibited a remarkable cytotoxic effect, while the polysaccharide extract from Spirulina sp. showed slight effect only at a higher concentration (2 mg/mL). All tested extracts were further investigated for improving immune cell killing ability and showed that Spirulina sp. polysaccharide extract was able to improve the immune cell killing ability. This extract was then investigated for its effects on the immune cell population, which demonstrated to have positive impact on NK cell population. To further explore the potential use, synergistic effect of Spirulina sp. polysaccharide extract with an already-in-use chemotherapeutic drug, gemcitabine, on immune cell cytotoxicity was investigated. The results showed that the immune cell cytotoxicity was enhanced in the co-treatment compared to the use of each treatment separately. The most apparent difference was observed in KKU055 cells where % living cells were reduced from 78.96% (immune cell alone) to 20.93% when the combined gemcitabine and Spirulina sp. extracts were used.

Extraction, purification, structural modification, activities and application of polysaccharides from different parts of mulberry.

The mulberry plant is a member of the Moraceae family and belongs to the Morus genus. Its entire body is a treasure, with mulberries, mulberry leaves, and mulberry branches all suitable for medicinal use. The main active ingredient in mulberries is mulberry polysaccharide. Studies have shown that polysaccharides from different parts of mulberry exhibit antioxidant, antidiabetic, antibacterial, anti-inflammatory, and blood pressure-lowering properties. There are more studies on the biological activities, extraction methods, and structural characterization of polysaccharides from different parts of mulberry. However, the structural characterization of mulberry polysaccharides is mostly confined to the types and proportions of monosaccharides and the molecular weights of polysaccharides, and there are fewer systematic studies on polysaccharides from different parts of mulberry. In order to better understand the bioactive structure of mulberry polysaccharides, this article discusses the recent research progress in the extraction, separation, purification, bioactivity, structural modification, and application of polysaccharides from different parts of mulberry (mulberry leaves, mulberry fruits, and mulberry branches). It also delves into the pharmacological mechanisms of action of mulberry polysaccharides to provide a theoretical basis for further research on mulberry polysaccharides with a view to their deeper application in the fields of feed and nutraceuticals.

Microwave‐Assisted Extraction of Polysaccharides From the Abelmoschus sagittifolius: Optimization, Structure, and Bioactivity

The aim of this study was to extract polysaccharides from Abelmoschus sagittifolius using the microwave‐assisted extraction (MAE) method. Three independent variables, including extraction temperature, microwave power, and microwave time, were chosen for investigation through single‐factor experiments. Response surface methodology (RSM) based on a Box‐Behnken design (BBD) was utilized to optimize the extraction conditions. The study found that the optimal conditions for polysaccharide extraction were a temperature of 63°C, a microwave power of 650 W, and a duration of 58 min. The polysaccharide yield under these conditions was 29.8 g/100 g, closely matching the experimental yield of 30.1 g/100 g. This yield was approximately 1.15 times higher than the yield achieved through conventional extraction methods (heat reflux extraction (HE) method). The study also demonstrated that polysaccharides extracted using the MAE method exhibited improved antioxidant capacity and enzyme inhibition and displayed significant inhibitory effects on HepG2 cells compared to the conventional extraction method. Moreover, the polysaccharides obtained from the optimized MAE process had a lower molecular weight and higher polydispersity than the HE method. These findings indicate that MAE is a promising, efficient, and environmentally friendly technology for extracting polysaccharides from A. sagittifolius.

Medicinal and edible polysaccharides from ancient plants: extraction, isolation, purification, structure, biological activity and market trends of sea buckthorn polysaccharides.

Sea buckthorn (Hippophae L.), a well-known medicinal and edible plant, is known as the "king of VC". Due to its excellent medicinal and nutritional value, it has been developed into a variety of functional products. Sea buckthorn polysaccharides (SPs), one of the important and representative active components, have attracted the attention of researchers in the fields of health food and medicine because of their potential beneficial effects on human health. Recently, SPs have shown various biological activities in in vitro and in vivo studies, such as anti-obesity, immunomodulatory, anti-tumor, antioxidant, anti-inflammatory, anti-fatigue, and hepatoprotective activities. This review provides a comprehensive and systematic summary of the extraction and purification methods, structural characterization, biological activity, and market trends of SPs to provide a theoretical basis for their therapeutic potential and sanitarian functions. A future scope is needed to further explore the medicinal and nutritional value of SPs and incorporate them in functional food products.

β(1,3) β(1,6) glucogalactan from Rhizopus microsporus var. oligosporus: extraction, characterization, antioxidant and α-amylase inhibitory activities.

In this study, the Box-Behnken experimental planning was used to optimize the extraction of polysaccharides from the cell wall of Rhizopus microspore var. oligosporus, with analysis of the quantitative effects of parameters pH, temperature and extraction time for polysaccharide yield. The optimal conditions for extraction were determined by the regression equation and evaluation of the response surface graphs, which indicated: pH 13, temperature of 120ºC and time of 60 min, with maximum yield around 18.5%. Fourier transform infrared spectroscopy analysis indicated typical polysaccharide signals. Nuclear magnetic resonance spectroscopy and monosaccharide analysis indicated a β(1,3) β(1,6) glucogalactan. The polysaccharide exhibited an average molecular weight of 120 kDa and a polymerization degree of 741. Antioxidant assays in vitro revealed the potential of polysaccharide in elimination of ABTS+ radical and hydroxyl radicals. EC50 values for free radical elimination were 7.69 and 17.8 mg/mL, for ABTS+ and hydroxyls, respectively. The polysaccharides showed potential for α-amylase inhibition with an EC50 of 1.66 mg/mL. The results suggest that β(1,3) β(1,6) glucogalactan from Rhizopus microsporus var. oligosporus can be used in biotechnological applications.

Ultrasound-assisted green and efficient extraction of polysaccharides from Chlorella vulgaris using deep eutectic solvents and improving their performance

An ultrasound-assisted DES was developed to efficiently and environmentally extract polysaccharides from Chlorella vulgaris (extraction efficiency 5.32 mg g−1). Chlorella extract has low cytotoxicity, good biocompatibility, and skin permeability.

Polysaccharide extraction from Myxopyrum smilacifolium trunk and its antioxidant capacity

As a traditional medicinal plant in Vietnam, Myxopyrum smilacifolium has been used for a long history to treat cough, nervous disorders, numbness, rheumatism, and cephalalgia. Nevertheless, reports on the antioxidant activity and extraction of M. smilacifolium polysaccharides are still very rare. This study was designed to extract a high yield of polysaccharides from the M. smilacifolium trunk and characterize it. As a result, the maximum yield of the polysaccharides of 5.13 ± 0.05% was obtained with an extraction time of three hours, extraction temperature of 100 oC, the ratio of water to sample of 1:50 as extraction solution, extraction number of times 3, and the ratio of ethanol to extract volume 5:1 (v/v). Polysaccharides was characterized by Fourier transform infrared spectroscopy (FT-IR) and high-performance gel-permeation chromatography (HPGPC). The average molecular weight of extracted polysaccharides was around 3.78 × 105 Da. In vitro assays dan impressive antioxidant activities of the extracted polysaccharides, containing 0.2423 ± 0.0028 mg GA/g or 0.2142 ± 0.0007 μmol AS/g. The IC50 values of polysaccharides in the DPPH and ABTS methods were 0.89 mg/mL and 3.85 mg/mL, respectively. These findings exhibited the potential for application or further research and development of polysaccharides from Myxopyrum smilacifolium.

Solution properties of laccase-treated pectic polysaccharides derived from steam-dried sugar beet pulp

Steam-drying of sugar beet pulp enables hot water extraction of pectic polysaccharides without adding acid to the extraction medium with a yield higher than the conventional preparation method but a lower molecular weight. The objective of the present study was to investigate the effect of laccase treatment on the molecular weight and solution properties of the pectic polysaccharides derived from steam-dried sugar beet pulp using hot water extraction. The weight average molecular weight and intrinsic viscosity of the pectic polysaccharides increased from 97.1 kDa to 147 kDa and from 0.383 dL/g to 0.525 dL/g due to isopropanol fractionation to remove low molecular weight fractions, respectively, and then to 661 kDa and 0.832 dL/g by the laccase treatment at the highest laccase rate examined, respectively. The intrinsic viscosity values showed a power-law relationship with the molecular weight, the exponent value of which was found to be 0.32 prior to the isopropanol fractionation or 0.38 after the isopropanol fractionation. These values were much lower than that derived theoretically for a random coil (0.5) and closer to that for a sphere (0.33). Compared to the results previously reported for various polysaccharides, the concentration dependence of the specific viscosity in the semi-dilute region was weaker, the critical concentration which is the threshold between dilute and semi-dilute solutions was higher, and the so-called coil overlap parameter was lower. The present results indicate that the pectic polysaccharides examined in this study had highly branched, compact, and spherical molecular structure.

Optimized Extraction and Fermentation of Antioxidant Polysaccharides from Polygonatum odoratum

Purpose: We wished to determine the optimal method for the extraction of polysaccharides with antioxidant activities from <i>Polygonatum odoratum</i>.Methods: Single-factor and orthogonal tests were applied to identify the best parameters for water extraction and fermentation. The solid-liquid ratio, temperature, and duration of water extraction were determined according to the total polysaccharide content. The fermentation temperature, agitation speed, inoculation amount, and fermentation time were investigated according to the optical density of Saccharomyces cerevisiae. The molecular weight distributions of water- and fermentation-extracted polysaccharides were determined by high performance gel permeation chromatography. The antioxidant activities of the polysaccharides were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ABTS radical scavenging capacities, and ferric reducing antioxidant power (FRAP).Results: The optimum water extraction parameters were 80°C for 2.5 h with a 1:60 solid-liquid ratio. For fermentation, an 8% extraction volume was inoculated with <i>S. cerevisiae</i>. Optimal fermentation occurred at 28°C for 28 h, with an agitation speed of 180 r/min. The molecular weights of water- and fermentation-extracted polysaccharides were 2.067×10<sup>4</sup> and 9.475×10<sup>3</sup> Da, respectively. The DPPH, ABTS, and FRAP results demonstrated an obvious increase in antioxidant activity after fermentation.Conclusion: The fermentation extraction of <i>P. odoratum</i> may provide valuable materials for future cosmetic use.

Extraction of Polysaccharides from Root of Pseudostellaria heterophylla (Miq.) Pax. and the Effects of Ultrasound Treatment on Its Properties and Antioxidant and Immune Activities.

The hydrophilic polysaccharides (PS) were isolated and purified from the tuberous roots of Pseudostellaria heterophylla. The extraction process of PS from Pesudostellariae radix was optimized by single-factor experiments and orthogonal design. The extract was purified by DEAE cellulose column to obtain the pure polysaccharide PHP. Then PHP was treated with different intensities of sonication to study the effect of sonication on PHP's characteristics and its biological activity in vitro and in vivo. The results of this study revealed that ultrasound treatment did not significantly change the properties of PHP. Further, with the increase of ultrasound intensity, PHP enhanced the proliferation and phagocytosis of macrophage RAW264.7. Meanwhile, it could also significantly improve the body's antioxidant activity and immune function. The results of this study demonstrated that PHP has the potential as a food additive with enhanced antioxidant and immune functions, and its biological activities could be enhanced by sonication.

Characterization and biological activities of polysaccharides extracted from Auricularia auricula with different extraction methods

Polysaccharides derived from Auricularia auricula exhibit diverse biological activities and hold significant potential for commercial utilization as functional food ingredients. In this investigation, polysaccharides from A. auricula were obtained using six extraction techniques (ammonium oxalate solution extraction, sodium hydroxide solution extraction, hot water extraction, pectinase and cellulase-assisted extraction, ultrasonic-assisted extraction, and microwave-assisted extraction). Subsequently, a comprehensive comparison was conducted to evaluate their physicochemical properties and biological functionalities. The ammonium oxalate solution extraction method yielded a higher extraction rate (11.76%) and polysaccharide content (84.12%), as well as a higher uronic acid content (10.13%). Although the six Auricularia polysaccharides had different molecular weight distributions, monosaccharide molar ratios, similar monosaccharide compositions, and characteristic functional groups of polysaccharides, they exhibited different surface morphology. In vitro assays showed that polysaccharides extracted by ammonium oxalate solution possessed good scavenging ability against DPPH free radical, hydroxyl free radical and superoxide anion free radical as well as reduction power of iron ion. At the same time, both polysaccharides extracted by ammonium oxalate solution and sodium hydroxide solution promoted NO production in mouse macrophages along with the secretion of cytokines TNF-α, IL-1β, and IL-6. These results indicated significant differences in the structure and characteristics among Auricularia polysaccharides prepared by various extraction methods, which may be related to the variety or origin of A. auricula; furthermore, their bioactivities varied accordingly in vitro assays where the ammonium oxalate solution extraction method was found more beneficial for obtaining high-quality bioactive Auricularia polysaccharides.

Antioxidant activity of Phellinus igniarius fermentation mycelia contributions of different solvent extractions and their inhibitory effect on α-amylase

Phellinus spp. have historically been used as traditional medicines to treat various diseases owing to their antioxidant, antitumor, and antidiabetic activities. Polysaccharides exhibit antidiabetic activity. In the present study, the polysaccharide contents of four Phellinus strains were compared. Phellinus igniarius QB72 possessed higher polysaccharide production, stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and α-amylase inhibitory activity. The three polysaccharides were sequentially extracted and partially purified from the fermentation mycelia using hot water, 1 % (NH4)2C2O4, and 1.25 M NaOH. Hot water extract polysaccharides exhibited higher DPPH radical scavenging and strong inhibitory activity against α-amylase with an IC50 value of 6.84 ± 0.37 mg/mL. The carbohydrate content of A1 (approximately 17457 Da) was approximately 88.28 %. The α-amylase inhibitory activity IC50 was decreased (3.178 ± 0.187 mg/mL) after DEAE water elution. P. igniarius QB72 hot-water extracts of partially purified polysaccharides have great potential as α-amylase inhibitors in food and medication-assisted additives.

Ultrasound-assisted extraction of polysaccharides from faba bean (Vicia faba L.) pod: optimization based on extraction yield, total phenolic content, and evaluation of its antioxidant activity

Ultrasound-assisted extraction of polysaccharides from faba bean (Vicia faba L.) pod: optimization based on extraction yield, total phenolic content, and evaluation of its antioxidant activity

Anti-inflammatory mechanism and application of mycelial polysaccharides

Studies have shown that mycelial polysaccharides can regulate the body's resistance to inflammation in different ways, this paper briefly describes how the extraction of mycelial polysaccharides can reduce the impact on the performance of mycelium, as well as some of the mycelial polysaccharides on the mechanism of inflammation resistance. In order to provide a theoretical basis for helping mycelial polysaccharides to become good anti-inflammatory drugs.

Ultrasonic extraction of Moringa oleifera seeds polysaccharides: Optimization, purification, and anti-inflammatory activities

Natural polysaccharides exhibit numerous beneficial properties, such as antioxidant, antitumor, hypoglycemic, and hypolipidemic activities. Moringa oleifera seeds are of high dietary and therapeutic value which drew a lot of attention. However, the regulation effect on anti-inflammatory activity of polysaccharides remains to be studied. Herein, novel bioactive polysaccharides (MOSP-1) were extracted from Moringa oleifera seeds, and the anti-inflammatory properties of MOSP-1 were uncovered. Ultrasound-assisted extraction (UAE) was used to prepare the polysaccharides with optimized conditions (70 °C, 43 min, and liquid-solid-ratio 15 mL/g). Then, DEAE-Sepharose Fast Flow columns were applied to isolate and purify MOSP-1. Rhamnose, arabinose, galactose, and glucose were identified as the monosaccharide constituents of MOSP-1, with a molecular weight of 5.697 kDa. Their proportion in molarity was 1:0.183:0.108:0.860 and 8 types of glycosidic linkages were discovered. Bioactive assays showed that MOSP-1 possessed scavenging activities against DPPH and ABTS radicals, confirming its potential antioxidation efficacy. In vitro experiments revealed that MOSP-1 could reduce the expression of inflammation-related cytokines, inhibit the activation of ERK, JNK, and p38 (the MAPK signaling pathway), and enhance phagocytic functions. This study indicates that polysaccharides (MOSP-1) from Moringa oleifera seeds with anti-inflammatory properties may be used for functional food and pharmaceutical product development.

Efficient extraction of pectic polysaccharides from thinned unripe kiwifruits by deep eutectic solvent-based methods: Chemical structures and bioactivities

To promote the potentially industrial applications of thinned unripe kiwifruits, two deep eutectic solvent-based methods, including deep eutectic solvent-assisted extraction (DAE) and microwave-assisted deep eutectic solvent extraction (MDE), were optimized for the extraction of polysaccharides from thinned unripe kiwifruits (YKP). Results showed that the yields of YKP-D prepared by DAE and YKP-DM prepared by MDE were extremely higher than YKP-H prepared by hot water extraction. Furthermore, YKP-H, YKP-D, and YKP-DM were mainly composed of pectic polysaccharides, including homogalacturonan (HG) and rhamnogalacturonan I (RG I) domains. Besides, both YKP-D and YKP-DM exhibited stronger antioxidant, anti-glycosylation, and immunomodulatory effects than those of YKP-H, and their higher contents of uronic acids and bound polyphenols as well as lower molecular weights could partially contribute to their bioactivities. Overall, these results revealed that the developed MDE method could be utilized as a promising method for highly efficient extraction of YKP with superior beneficial effects.

Effects of Near-Freezing Temperature Combined with Jujube Polysaccharides Treatment on Proteomic Analysis of 'Diaogan' Apricot (Prunus armeniaca L.).

This study involved the extraction of polysaccharides from jujube for application in apricot storage. Although near-freezing temperature (NFT) storage is commonly employed for preserving fresh fruit, its effectiveness is somewhat limited. Incorporating jujube polysaccharides was proposed to augment the preservative effect on apricots. Our findings demonstrated that the combined use of NFT and jujube polysaccharides can maintain fruit color, and effectively inhibit decay. Additionally, Tandem Mass Tag (TMT) quantitative proteomic technology was utilized to analyze protein variations in 'Diaogan' apricots during storage. This dual approach not only markedly lowered the activity of polyphenol cell wall-degrading enzymes (p < 0.05) but also revealed 1054 differentially expressed proteins (DEPs), which are related to sugar and energy metabolism, stress response and defense, lipid metabolism, and cell wall degradation. The changes in DEPs indicated that the combined use of NFT and jujube polysaccharides could accelerate the conversion of malic acid to oxaloacetic acid and regulate antioxidant ability, potentially extending the storage lifespan of apricot fruit.

Использование новых кормовых добавок из местных растительных ресурсов в рационах кур-несушек

Purpose. To establish the effect of extruded grain of amaranth (Amaranthus hybridus chlorostachys) with and without the addition of a complex of enzymes, sunflower polysaccharide extract (hereinafter referred to as SPE), sunflower and pumpkin fuses in combination with bischofite on economic, biological, hematological indicators and the immune status of laying hens. Materials and Methods. The experiment was carried out on laying hens of the industrial herd of the Hayseks brown cross at the breeding enterprise of the Volgograd region of the Svetloyarsky district on the basis of Agricultural enterprise "Svetly" JSC "Agrofirm Vostok". Blood biochemical parameters were determined according to the methods of JSC "Deacon-DS" using the appropriate set of biochemical reagents on automatic biochemical analyzers URIT-800Vet, URIT-3020, other studies – on certified equipment in the complex analytical laboratory of VRIMMP (Volgograd, Russia). The obtained results were processed using software, calculating the mean value (M), standard errors of the mean value (±SEM) and determination of the Student-Fisher test for the significance of the difference. Results. The positive dynamics of cholesterol reduction by 6.87% was proved with the addition of 10% amaranth and by 9.86% with the addition of 10% amaranth and 250 g/t of the multienzyme complex. The use of sunflower polysaccharide extract in feeding laying hens – a by-product of sunflower oil production – in the amount of 3; 5 and 7% contributed to an increase in the yield of hatching eggs, respectively, by 88, 239 and 149 pcs. regarding control, increase in the intensity of egg production, feed conversion. The highest efficiency in the production of hatching eggs was achieved when using sunflower polysaccharide extract at a dosage of 5.0% in the diet structure. A significant increase in the productivity of chickens of all experimental groups by a value of 2.00 to 3.94% (P≤0.001) was established with the addition of pumpkin, sunflower phosphatides and bischofite, while the upper value was recorded with the addition of sunflower phosphatides and bischofite. The addition of pumpkin phosphatides contributed to the greatest increase in the weight of the laid egg relative to the control by 2.7 g (4.38%). Feeding both types of phosphatides contributed to the economy of feed, lowering cholesterol levels by 2 to 3.6%. The use of bischofite was manifested in a significant increase in the thickness of the shell, respectively, by a value from 13.0 to 21.2 microns. Conclusion. The greatest reduction in cholesterol levels (by 9.86%) and efficiency in the production of eggs was achieved with the addition of 10% amaranth and 250 g/t of the multi-enzyme complex. The best economic and biological indicators of poultry were recorded when using sunflower polysaccharide extract at a dosage of 5.0% in the diet structure. The addition of sunflower phosphatides in the amount of 3% and 2.6% bischofite contributed to a significant increase in the productivity of chickens by 3.94% (P≤0.001). The addition of pumpkin phosphatides contributed to the greatest increase in the weight of the laid egg relative to the control by 2.7 g (4.38%). The inclusion of bischofite in the diet of laying hens resulted in a significant increase in shell thickness by 13.0 to 21.2 µm.