Lactoferrin(LF)is a non-heme iron-binding glycoprotein mainly found in mammalian milk,with various biological functions including,antibacterial anti-inflammatory,antiviral,and iron binding.LF is widely used in the food and pharmaceutical industries.Currently,the LF market is dominated by bovine lactoferrin(BLF),with a relatively limited source.With the rapid development of the goat milk industry in China and the increasing demand for functional foods,the goat milk deep processing industry urgently needs to be developed.Studies have shown that goat lactoferrin(GLF)has multiple biological functions and has a glycosylation pattern similar to that of human lactoferrin,and it is a high-quality LF resource.However,the development and utilization of GLF are seriously inadequate.Promoting the development and utilization of GLF is of positive significance for enriching the LF market and promoting the development of the goat milk deep processing industry.In addition,heterologous lactoferrin is mainly ingested orally,and upon entering the intestine.LF relies on its unique protein structure to exert various biological functions such as regulating intestinal microbiota and improving intestinal inflammation,which has positive significance in helping to establish and maintain a healthy intestinal environment.However,lactoferrin is easily digested by pepsin.For infants and young children with immature digestive systems,lactoferrin can still enter the intestine in its complete form.However,for adults with fully established digestive systems,lactoferrin is hydrolyzed during the gastric digestion stage,losing its biological activity as a complete protein.To achieve a wider application of lactoferrin in adult nutritional supplements,it can be delivered to facilitate its passage through the gastric digestion stage and delivery to the intestinal tract in the form of intact protein,thereby enhancing its biological efficacy value.In this study,GLF was obtained from goat colostrum freeze-dried powder by pretreatment,separation,and purification,and its protein identification,basic structure,and properties were determined,providing a reference for the development of GLF.This study also proposed a series of new methods to construct p H responsive intestinal delivery carriers based on sodium alginate(SA)and carboxymethyl chitosan(CMCS),and evaluated the GLF load and potential intestinal delivery performance of each delivery carrier,providing guidance for achieving better use of functional goat lactoferrin by adults.The main research results are as follows:(1)Study on the isolation,purification,basic structure,and properties of goat lactoferrinGoat lactoferrin(GLF)was isolated and purified from goat colostrum freeze-dried powder by resuspension,defatting,and removal of casein.SP Sepharose Fast Flow and CM Sepharose Fast Flow were used as the cation exchange resins for separation and purification,and the final LF obtained accounted for 97.67 ± 0.34% of the total protein content,with a total yield of89.96± 0.95%.The LF was identified as goat lactoferrin by peptide mass fingerprinting,with a relative molecular weight of approximately 81,800 Da,and its secondary structure composition was not significantly different from that of bovine lactoferrin(BLF).Compared with BLF(16%),GLF had a higher iron saturation of 28%,making it a good source of iron supplement.A simulated digestion experiment showed that GLF was unstable in the stomach and was completely hydrolyzed by pepsin in only 20 minutes,indicating the necessity of delivering GLF to improve its gastric digestion retention rate.(2)p H-driven carboxymethyl chitosan self-coacervation as a strategy for encapsulation and delivery of goat lactoferrinTo improve the retention rate of GLF in the stomach,this study used the properties of CMCS amphiphilic polymer to promote the formation of CMCS self-coacervates by adjusting the p H and applied it to the delivery of GLF.The highest yield of CMCS self-coacervates was obtained at the isoelectric point p H of 4.8,which was 71.35 ± 1.34%.Electrostatic interactions,hydrogen bonds,and hydrophobic interactions promoted the formation of this self-coacervate.CMCS self-coacervates exhibited good p H responsiveness and protein delivery performance.After 2 hours of simulated stomach digestion,the retention rate of GLF was 75.61 ± 3.13%.In simulated intestinal fluid(SIF),GLF could be completely released within 15 minutes by the rapid disintegration of CMCS self-aggregates.(3)Hydrogen-bond-driven preparation of sodium alginate/carboxymethyl chitosan noncrosslinked hydrogel beads for goat lactoferrin deliveryA hydrogen-bond-driven method for preparing p H-responsive sodium alginate/carboxymethyl chitosan(SA/CMCS)non-crosslinked hydrogel beads was developed based on the gelation properties of SA under acidic conditions and the amphiphilic properties of CMCS.Non-crosslinked SA/CMCS hydrogel beads were successfully prepared when the crosslinking solution p H was less than 3.0.Fluorescence labeling results showed that the hydrogel beads had a "shell-core" structure and that CMCS was mainly distributed on the outer layer of the hydrogel beads.The hydrogel beads exhibited good p H responsiveness and GLF loading capacity,with the highest encapsulation efficiency and loading capacity of GLF obtained from hydrogel beads prepared under p H 1.5 conditions,which were 95.68 ± 0.31%and 42.65 ± 0.21%,respectively.The hydrogel beads improved the retention rate of GLF during simulated stomach digestion to 77.72 ± 2.74% and could completely release GLF within 2 hours as the hydrogel beads gradually disintegrated in simulated intestinal fluid.(4)Citric acid crosslinked sodium alginate/carboxymethyl chitosan hydrogel beads for goat lactoferrin deliveryTo further enhance the retention of GLF during stomach digestion,citric acid(CA)was used as the crosslinking agent to prepare p H-responsive hydrogel beads of sodium alginate/carboxymethyl chitosan(SA/CMCS)with different swelling properties by changing the ratio of SA and CMCS.The swelling ratio of the SA/CMCS hydrogel beads in gastric acid p H environment decreased as the SA content increased.The S2C1 hydrogel beads prepared with a volume ratio of 2:1 of SA to CMCS had the highest encapsulation efficiency and loading capacity of GLF,which were 96.96 ± 2.31% and 26.70 ± 1.34%,respectively.These hydrogel beads improved the retention rate of GLF during simulated stomach digestion to 82.15 ± 2.13%.The loading and release processes did not affect the structure of GLF.The release kinetics indicated that the release of GLF resulted from the combined effects of hydrogel bead dissolution,swelling,GLF diffusion,and electrostatic interactions.(5)The influence of chitosan on the performance of citric acid crosslinked sodium alginate/carboxymethyl chitosan hydrogel beads for goat lactoferrin deliveryTo reduce the swelling of SA/CMCS hydrogel beads in gastric acid conditions and further improve the retention of GLF during simulated stomach digestion,chitosan(CS)was introduced into the CA crosslinked SA/CMCS hydrogel system to prepare CS/SA/CMCS hydrogel beads.Fluorescence labeling results showed that CS was coated on the outer layer of the hydrogel beads.The introduction of CS not only improved the thermal stability of the hydrogel beads but also reduced their swelling ratio in simulated gastric acid conditions.Among them,CS/S2C1 had the smallest swelling ratio in the simulated gastric acid environment(only0.32)and improved the retention rate of GLF during simulated stomach digestion to 90.14 ±1.96 %.The CS/SA/CMCS hydrogel beads also maintained the stability of the protein structure during the loading and release of GLF.(6)Study on the cytotoxicity and stability of goat lactoferrin delivery carriersFinally,cytotoxicity and stability experiments were carried out on the four p H-responsive intestinal delivery systems based on natural polysaccharides prepared in this study.The results showed that all four delivery systems prepared in this study were non-toxic to cells and had a good temperature,humidity,and light stability,indicating that the various delivery systems prepared in this study have potential practical application value and suitable delivery carriers can be selected according to the actual application scenarios. |