Improvement of amphipathic properties with molecular structure unfolding and activation of cottonseed protein as ultra stable and safe emulsifier by deamidation.

By-product cottonseed proteins are excellent options for numerous applications due to their superior properties and lower cost. However, its complex folded structure and large molecular weight lead to lower reactivity and insufficient amphiphilicity. Cottonseed protein isolate (CPI) is less-soluble in water. Therefore, we improved the amphiphilicity of CPI with associated hydrolysis, molecular structure unfolding, and activation by alkaline-induced deamidation (at 24, 36, and 72 h) and produced three cottonseed protein hydrolysates CPH 24, 36, and 72. FTIR/UV-CD measurements confirmed the conformational changes and conversion of the structural content. Particle size decreased 2503.4-771.8 nm, while surface hydrophobicity (133.5-326.7), carboxyl content (1.13 × 10Ö¾3-2.09 × 10Ö¾3), and flexibility increased, signifying hydrolysis, unfolding, and amphiphilicity improvement. Longer deamidation (CPH 72) exhibited the best properties, its prepared emulsions were long-term stable under all the environmental stresses without visible phase separation after at least 40 days of storage except at pH 4. Compared to CPI, it had smaller droplets (939.3-264.9 nm) and larger absolute ζ-potential (-26.5 to -58.0 mV). From the in-vitro cytotoxicity test, deamidated CPI is extremely safer than commonly used synthetic surfactants. This research provides a new method for producing multifunctional emulsifiers from CPI, which could be utilized in the development of functional foods/non-foods.

Low fish meal diet supplemented with probiotics ameliorates intestinal barrier and immunological function of Macrobrachium rosenbergii via the targeted modulation of gut microbes and derived secondary metabolites.

The unsuitable substitution ratio of fish meal by plant protein will reshape the intestinal microbial composition and intestine immunity. However, previous studies were mostly limited to investigating how different feed or probiotics characterized the microbial composition but ignored the biological interactions between bacteria and host physiology through secondary metabolites. Therefore, this study integrates the apparent indicators monitoring, 16S rDNA sequencing, and metabonomics to systematically investigate the effects of cottonseed protein concentrate (CPC) substitution of fish meal and Bacillus coagulans intervention on gut microbes, secondary metabolites, and intestinal immunity of Macrobrachium rosenbergii. Prawns were fed with three diets for 70 days: HF diets contained 25% fish meal, CPC in LF diets were replaced with 10% fish meal, and LF diets supplemented with 2 × 108 CFU/g diet B. coagulans were designated as BC diets. Results showed that CPC substitution induced a significant decrease in digestive enzyme activities (trypsin and lipase) and gut barrier protein PT-1 expression and a significant increase in γ-GT enzyme activity and inflammatory-related factors (Relish and Toll) expression. B. coagulans treatment mitigated the negative changes of the above indicators. Meanwhile, it significantly improved the expression levels of the barrier factor PT-1, the reparative cytokine IL-22, and Cu/Zn-SOD. CPC substitution resulted in a remarkable downregulated abundance of Firmicutes phyla, Flavobacterium spp., and Bacillus spp. B. coagulans treatment induced the callback of Firmicutes abundance and improved the relative abundance of Sphingomonas, Bacillus, and Ralstonia. Functional prediction indicated that CPC substitution resulted in elevated potential pathogenicity of microbial flora, and B. coagulans reduces the pathogenesis risk. Pearson's correlation analysis established a significant positive correlation between differential genera (Sphingomonas, Bacillus, and Ralstonia) and secondary metabolites (including sphingosine, dehydrophytosphingosine, amino acid metabolites, etc.). Meanwhile, the latter were significantly associated with intestinal immunoregulation-related genes (Cu/Zn-SOD, IL-22, PT-1, Toll, and Relish). This study indicated that B. coagulans could mediate specific gut microbes and the combined action of multiple functional secondary metabolites to affect intestinal barrier function, digestion, and inflammation. Our study revealed the decisive role of gut microbes and derived secondary metabolites in the model of dietary composition-induced intestinal injury and probiotic treatment from a new perspective.

Largemouth bass (Micropterus salmoides) exhibited better growth potential after adaptation to dietary cottonseed protein concentrate inclusion but experienced higher inflammatory risk during bacterial infection.

Cottonseed protein concentrate (CPC) has been proven to partially replace fishmeal without adverse effects on fish growth performance, while little information is known about the effects on liver health during bacterial infection. In the present study, 15% CPC was included into the diet of juvenile largemouth bass (32.12 ± 0.09g) to replace fishmeal for 8 weeks, with fish growth potential and hepatic inflammatory responses during Nocardia seriolae (N. seriolae) infection systemically evaluated. After adaptation to dietary CPC inclusion, largemouth bass even exhibited better growth potential with higher SGR and WGR during the last three weeks of whole feeding trial, which was accompanied with higher phosphorylation level of TOR signaling and higher mRNA expression level of myogenin (myog). At the end of 8-weeks feeding trial, the histological structure of largemouth bass liver was not significantly affected by dietary CPC inclusion, accompanied with the similar expression level of genes involved in innate and adaptive immunity and comparable abundance of T cells in bass liver. N.seriolae infection induced the pathological changes of bass liver, while such hepatic changes were more serious in CPC group than that in FM group. Additionally, RT-qPCR results also suggested that largemouth bass fed with CPC experienced much higher inflammatory potential both in liver and gill during N. seriolae infection, which was accompanied with higher expression level of genes involved in pyroptosis. Therefore, this study demonstrated that the application of CPC in largemouth bass diet should be careful, which may induce higher inflammatory potential during N. seriolae infection.

Effects of replacing fish meal with cottonseed protein concentrate on the growth, immune responses, digestive ability and intestinal microbial flora in Litopenaeus vannamei.

The effects of cottonseed protein concentrate (CPC) in place of fishmeal on the growth performance, immune response, digestive ability and intestinal microbiota of Litopenaeus vannamei were investigated in this study. L. vannamei (initial body weight: 0.42 ± 0.01g) was fed for 8 weeks by four isonitrogenous and isolipid feeds with CPC replacing fishmeal (FM) at 0% (control), 15% (CPC15), 30% (CPC30) and 45% (CPC45), respectively. At the end of the study, the final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR) and protein efficiency ratio (PER) of L. vannamei in CPC15 and CPC30 groups were significantly increased, while the feed conversion ratio (FCR) of L. vannamei in the CPC30 group was significantly reduced when compared with the FM group (P < 0.05). After Vibrio parahaemolyticus infection, the cumulative mortality of L. vannamei in CPC15 within 24 hpi was significantly lower than that of the control group (P < 0.05). When compared with the control group, the activities and expression of the immunity-related enzymes in the hepatopancreas had almost the same obvious change trend in the CPC-containing groups, which indicated that the replacement for fishmeal by CPC led to significant immune response in L. vannamei. Besides, significant up-regulation of the digestive enzyme activities were observed in the CPC-containing groups. Analysis of intestinal microbiota showed that significant difference in alpha diversity existed between the CPC-containing groups and the control group. The relative abundances of several top 10 dominated species at the phylum and genus levels were significantly changed in the CPC-containing groups compared with the control group (P < 0.05). Functional prediction of the microbiota indicated that the pathway of protein digestion and absorption was significantly more abundant while the pathways of nitrotoluene degradation, aminobenzoate degradation, atrazine degradation, dioxin degradation and xylene degradation were significantly less abundant in the CPC-containing groups than the FM group (P < 0.05). In summary, optimal dietary CPC replacement of FM could improve the growth, immunity, digestive capacity and the diversities of the intestinal microbial flora of L. vannamei. However, parts of the functions of the intestinal microbial flora were decline.

Effects of replacing fishmeal with cottonseed protein concentrate on growth performance, blood metabolites, and the intestinal health of juvenile rainbow trout (Oncorhynchus mykiss).

Cottonseed protein concentrate (CPC) is a potential non-food protein source for fishmeal replacement in fish feed. However, a high inclusion level of CPC in diets may have adverse effects on the metabolism and health of carnivorous fish. This study aimed to investigate CPC as a fishmeal alternative in the diet of rainbow trout Oncorhynchus mykiss based on growth performance, blood metabolites, and intestinal health. Five isonitrogenous (46% crude protein) and isolipidic (16% crude lipid) diets were formulated: a control diet (30% fishmeal) and four experimental diets with substitution of fishmeal by CPC at 25%, 50%, 75%, and 100%. A total of 600 fish (mean body weight 11.24g) were hand-fed the five formulated diets to apparent satiation for eight weeks. The results showed no adverse effects on growth performance when 75% dietary fishmeal was replaced by CPC. However, reduced growth and feed intake were observed in rainbow trout fed a fishmeal-free diet based on CPC (CPC100%). Changes in serum metabolites were also observed in CPC100% compared with the control group, including an increase in alanine aminotransferase (ALT), a decrease in alkaline phosphatase (ALP), alterations in free amino acids, and reductions in cholesterol metabolism. In addition, the CPC-based diet resulted in reduced intestinal trypsin, decreased villus height and width in the distal intestine, upregulated mRNA expression levels of inflammatory cytokines in the intestine, and impaired gut microbiota with reduced bacterial diversity and decreased abundance of Bacillaceae compared with the control group. The findings suggest that the optimum substitution rate of dietary fishmeal by CPC for rainbow trout should be less than 75%.

Effects of hydrolyzed cottonseed protein supplementation on performance, blood metabolites, gastrointestinal development, and intestinal microbial colonization in neonatal calves.

The objective of this study was to investigate the effects of an enzymatically hydrolyzed cottonseed protein (HCSP) as a peptide source on performance, blood metabolites, gastrointestinal development, and intestinal microbes. Forty-eight newborn Holstein calves were randomly assigned to 1 of the 4 dietary treatments including 0, 2, 4, and 6% of HCSP (dry matter basis). All calves received the same amount of pasteurized whole milk, weaned on d 56 of the experiment, and the study was concluded on d 70. Data were analyzed using PROC MIXED in SAS (SAS Institute Inc., Cary, NC) as a randomized complete block design with linear and quadratic contrasts. Results showed that increased amount of HCSP linearly decreased the starter intake during the postweaning (d 57 to 70) and overall period (d 1 to 70). In addition, when dietary HCSP increased during the overall period, average daily gain tended to linearly decrease. All skeletal growth variables also linearly decreased as dietary HCSP increased at the end of the study, except for body length, which did not differ among the treatments. Serum cortisol concentration was higher in calves supplemented with 6% of HCSP at weaning and at the end of the study. This indicates that these calves may have experienced a stressful condition compared with calves in other treatments. Total antioxidant capacity was quadratically affected by HCSP supplementation; calves fed 2 and 4% of HCSP diets had the highest total antioxidant capacity, whereas calves fed 0 and 6% HCSP diets had lower total antioxidant capacity at weaning and at end of the study. Calves supplemented with 6% HCSP had lower empty reticulo-rumen and omasum weights and rumen wall thickness compared with calves in other treatments at the end of the study. In conclusion, supplementation of HCSP at the rate of 2% of starter diet enhanced antioxidant status without any detrimental effects on the performance and metabolic status of calves, whereas greater inclusion rates impaired starter intake and growth of calves, and exposed them to a stressful status.

Effect of enzymatic hydrolysate of cottonseed protein supplementation on growth performance and intestinal health of nursery pigs in Thailand.

This study investigated the effects of enzymatic hydrolysate of cottonseed protein (EHCP) supplementation on the growth performance and intestinal health of nursery pigs in Thailand. A total of 180 newly weaned piglets were randomly allocated to 3 groups with 6 replicates in each group and 10 piglets per replicate. Nursery pigs were fed three diets containing 0, 1%, and 1.5% EHCP for 28-63 days of age. The results indicated that 1% EHCP supplementation increased average daily feed intake (ADFI) and average daily gain (ADG) and decreased feed conversion rate (FCR) in the numerical, suggesting that appropriate EHCP supplementation could numerically improve growth performance of nursery pigs in Thailand. Moreover, 1% EHCP supplementation significantly decreased intestinal crypt depth and diarrhea incidence and increased intestinal villus height to crypt depth ratio and fecal consistency, suggesting that optimum EHCP supplementation could improve intestinal morphology and decreased diarrhea incidence of nursery pigs in Thailand. Furthermore, 1% EHCP supplementation significantly improved intestinal glutathione (GSH) level and superoxide dismutase (SOD) activity and indicated that optimal EHCP supplementation could improve intestinal antioxidant capacity of nursery pigs in Thailand. Optimum EHCP supplementation numerically increased growth, significantly decreased diarrhea incidence, significantly improved intestinal morphology and antioxidant capacity of nursery pig in Thailand.

Cottonseed protein concentrate (CPC) suppresses immune function in different intestinal segments of hybrid grouper ♀Epinephelus fuscoguttatus×♂Epinephelus lanceolatu via TLR-2/MyD88 signaling pathways.

Cottonseed protein concentrate (CPC) has similar amino acid composition compared with fish meal, and has the characteristics of low gossypol and low toxicity. The present study was conducted to investigate the growth performance, antioxidant capacity and different intestinal segments immune responses of hybrid grouper to replacement dietary fish meal ofCPC. Six iso-nitrogenous (50% crude protein) and iso-lipidic (10% crude lipid) diets were formulated: a reference diet (FM) containing 60% fishmeal and five experimental diets (12%, 24%, 36%, 48 and 60%) in which fishmeal protein was substituted at different levels by CPC to feed fish (initial body weight: 11 ±â€¯0.23 g) for 8 weeks. Thena challenge test with injection of Vibrio parahaemolyticus was conducted for 7 days until the fish stabilized. The results showed that specific growth rate (SGR) was the highest with 24% replacement level and feed conversion ratio (FCR)was significantly increased when the replacement level reached 48% (P < 0.05). The content of malonaldehyde (MDA) in the serum was significantly increased when the replacement level reached 36% (P < 0.05). The plica height in the proximal, mid and distal intestine were significantly decreased with the replacement level up to 48% (P < 0.05). Hepatic fat deposition wasaggravatedwhen the replacement level reached 36% (P < 0.05). The expression of IL-6, TNF-α, and IL-1ß mRNAs were significantly up-regulated (P < 0.05). The hepcidin mRNA expression was significantly down-regulated (P < 0.05). In proximal intestine (PI) and mid intestine (MI), IFN-γ mRNA expression was significantly up-regulated (P < 0.05). These results suggested that the CPC decreased hybrid grouper growth performance and inflammation function, and different inflammation function responses in PI,MI, and distal intestine (DI) were mediated partly by the TLR-2/MyD88 signaling pathway. According to the analysis of specific growth rate, the dietary optimum replacement level and maximum replacement level were estimated to be 17% and 34%, respectively.