Supercritical fluid extract from maca alleviates colitis induced by dextran sulfate sodium in mice

The objective of this study was to investigate the possible protective effects of maca (Lepidium meyenii) extract (MLE) by supercritical fluid extraction on dextran sodium sulfate (DSS)-induced colitis. Experimental colitis was induced by giving male BALB/c mice 3% DSS in drinking water, and MLE (30 mg/kg BW), sulfasalazine (100 mg/kg BW) or vehicle were administered orally. DSS challenge caused significant body weight loss, rectal bleeding, diarrhea, shortened colon length, histological changes, and increased myeloperoxidase (MPO) activity in DSS-treated mice. Oral administration of MLE significantly relieved the symptoms of diarrhea and rectal bleeding, and reduced colonic MPO activity (p<0.05). MLE treatment inhibited expression of several colonic proteins related to inflammatory responses, such as interleukin (IL)-1β, tumor necrosis factor-α, IL-6, and S100 calcium-binding protein A8, whose expressions were increased significantly by DSS treatment. These results suggest that MLE can alleviate DSS-induced colitis in mice by modulating colonic inflammatory mediators.     

Lactobacillus rhamnosus alleviates intestinal barrier dysfunction in part by increasing expression of zonula occludens-1 and myosin light-chain kinase in vivo

The effects of lactobacilli on impaired intestinal barrier function and paracellular permeability were evaluated in human epithelial Caco-2 cells treated with tumor necrosis factor-α and in mice with colitis induced by dextran sodium sulfate (DSS). Filter-grown Caco-2 monolayers were used as the intestinal epithelial model. Among the 4 lactobacilli studied, Lactobacillus rhamnosus OLL2838 most effectively suppressed barrier impairment and increased IL-8 secretion induced by tumor necrosis factor-α in Caco-2 cells; however, the conditioned medium from OLL2838 did not show any effect on barrier functions. The in vivo effects of OLL2838 on intestinal epithelial barrier function and colonic inflammation were assessed in DSS-induced colitis of BALB/c mice. Oral treatment with both live and heat-killed OLL2838 suppressed weight loss and recovered colon length. Additionally, barrier function was restored by the administration of live and heat-killed OLL2838 to the DSS-treated animals, which conferred protection against the increase in mucosal permeability associated with DSS-induced colitis. This may at least partially be because of the increased expression of zonula occludens-1 (4.8-fold) and myosin light-chain kinase (3.1-fold) in intestinal epithelial cells isolated from mice of the heat-killed OLL2838 group. Therefore, L. rhamnosus OLL2838 would be useful in the treatment of gastrointestinal diseases such as inflammatory bowel disease.     

Prato cheese containing Lactobacillus casei 01 fails to prevent dextran sodium sulphate-induced colitis

The role of experimental probiotic Prato cheese containing Lactobacillus casei 01 in the prevention of dextran sodium sulphate (DSS)-induced ulcerative colitis in mice was evaluated. For the DSS in vivo model, mice were divided into six groups. Groups 1–3 represented noninflamed groups and groups 4–6 received a DSS (2%) solution. Mice from groups 1 and 4 intragastrically received 500 μL of phosphate-buffered saline (control groups), while mice from groups 2 and 5 intragastrically received 500 μL of conventional Prato cheese (control groups). Finally, mice from group 3 and 6 intragastrically received 500 μL of Prato probiotic cheese containing L. casei 01 (9.47 log cfu mL⁻¹). Groups treated with probiotic Prato cheese exhibited reduced weight loss caused by the consumption of DSS solution. However, the probiotic cheese failed to reduce the inflammation scores in DSS-induced colitis group. Overall, probiotic Prato cheese was not effective to ameliorate the symptoms of DSS-induced colitis.     

表没食子儿茶素没食子酸酯对葡聚糖硫酸钠诱导小鼠炎症性肠病的保护作用

本研究旨在探讨表没食子儿茶素没食子酸酯（Epigallocatechin-3-gallate,EGCG）对葡聚糖硫酸钠（Dextran sulfate sodium,DSS）诱导的小鼠结肠炎和肠道菌群的影响。将C57BL/6雄性小鼠分为正常对照组、肠炎模型组和EGCG处理组（50 mg/kg）,每组10只,连续灌胃给药9 d。通过称取小鼠体质量,观察并记录小鼠大便黏稠度、大便出血情况,测量小鼠结肠长度和检测血清中炎症因子来评估EGCG对DSS诱导的小鼠结肠炎症的改善作用;通过分析结肠病理形态、紧密连接蛋白的表达量、肠道菌群多样性和肠道菌群结构来评估EGCG对DSS诱导的结肠炎小鼠肠道菌群的影响。结果表明,EGCG能够有效改善DSS诱导的结肠炎小鼠体质量的下降、腹泻、便血、结肠缩短等不良反应;缓解DSS诱导的小鼠结肠炎导致的全身性慢性炎症和肠道屏障损伤;改善DSS诱导的结肠炎小鼠肠道菌群紊乱,恢复肠道菌群多样性,降低厚壁菌门的相对丰度,提高拟杆菌门的相对丰度,促进有益菌Akkermansia、Alistipes和Bacteroides的增殖并抑制有害菌Desulfovibrio、Escherichia-Shigella和Helicobacter的生长。因此,EGCG通过保护肠道屏障和调节肠道菌群紊乱,从而有效改善DSS诱导的小鼠结肠炎症。     

芹菜苷对葡聚糖硫酸钠诱导小鼠结肠炎的缓解作用

目的：借助葡聚糖硫酸钠（dextran sulfate sodium,DSS）诱导的结肠炎小鼠模型,探究芹菜苷对肠道健康的影响。方法：将30 只雄性C57BL/6J小鼠随机分成正常组、模型组、芹菜苷组。正常组和模型组全程给予AIN93G纯饲料,芹菜苷组全程给予基于AIN93G纯饲料配方调整的芹菜苷（质量分数0.016 7%）定制饲料,饲喂7 d后,模型组和芹菜苷组的饮用水更换为含质量分数3% DSS的无菌水进行肠炎造模,造模6 d后,停止给予DSS无菌水,24 h后处死小鼠;每天观察并记录小鼠饮食量、体质量,计算小鼠疾病活动指数（disease activity index,DAI）,在实验结束后测定小鼠脾脏指数,血清中肿瘤坏死因子（tumor necrosis factor,TNF）-α、白细胞介素（interleukin,IL）-6水平及结肠组织中髓过氧化物酶（myeloperoxidase,MPO）、诱导型一氧化氮合酶（inducable nitric oxide synthase,iNOS）活力,观察小鼠结肠病理及杯状细胞的数量状况。结果：与模型组相比,芹菜苷可显著缓解结肠炎小鼠的体质量下降（P＜0.05）,降低DAI（P＜0.001）与脾脏指数（P＜0.001）以及血清中炎症因子TNF-α（P＜0.001）与IL-6（P＜0.01）的质量浓度;另外,芹菜苷对结肠缩短有极显著改善作用（P＜0.01）,并可减轻结肠炎小鼠结肠组织损伤和降低炎症浸润水平,极显著降低MPO及iNOS活力（P＜0.001）,显著增加杯状细胞的数量（P＜0.05）,促进黏液分泌,维持肠道屏障功能完整。结论：25 mg/kg mb的芹菜苷剂量可缓解DSS诱导的小鼠结肠炎。     

Lactobacillus plantarum CAU1055 ameliorates inflammation in lipopolysaccharide-induced RAW264.7 cells and a dextran sulfate sodium–induced colitis animal model

This study aimed to screen lactic acid bacteria (LAB) for their anti-inflammatory activity by using RAW264.7 cells and dextran sulfate sodium (DSS)-induced colitis. In all, 192 LAB strains were isolated from healthy human feces, of which 8 strains showed excellent nitric oxide (NO) inhibitory activity. Peptidoglycan extracts of these 8 LAB strains were subjected to NO assay, Western blot, and ELISA. Among the 8 tested strains, extracts of 4 strains significantly inhibited the production of NO, related enzyme activities such as inducible nitric oxide synthase and cyclooxygenase 2, and key cytokines such as tumor necrosis factor-α and IL-6 in RAW264.7 cells. The 4 strains belonged to Lactobacillus (CAU1054, CAU1055, CAU1064, and CAU1301). Oral administration of the 4 strains inhibited DSS-induced body weight loss, colon shortening, and colon damage in ICR mice. The colon tissue of the mice treated with Lactobacillus plantarum strain CAU1055 had significantly reduced levels of inducible nitric oxide synthase, cyclooxygenase 2, tumor necrosis factor-α, and IL-6. We found that strain CAU1055 could be used as a candidate probiotic strain for the prevention and treatment of inflammatory bowel disease. Further studies are warranted to confirm the mechanisms of interaction between peptidoglycan of L. plantarum strain CAU1055 and upstream cellular signaling mediators.     

Heat-resistant structural features of bovine β-lactoglobulin A revealed by NMR H/D exchange observations

The thermal unfolding of the A variant of bovine β-lactoglobulin (β-Lg), in 50 mm phosphate buffer at pH 3.0, was followed by nuclear magnetic resonance (NMR) hydrogen/deuterium (H/D) exchange observations. The exchange behaviour of a large number of backbone amide protons (H_Ns) was monitored at temperatures between 37°C and 80°C to determine the relative thermal stability of different elements of the native protein structure. The H/D exchange was rapid at 37°C for the H_Ns on the residues in loops and in the terminal regions of the native protein. Further H/D exchange was promoted by heat treatment at temperatures up to 80°C and was completed for the identifiable H_N of the various α and β structural elements in the following order: D–E strand (55–60°C); C–D strand and α-helix (60–65°C); A–B, A–I and E–F strands (65–70°C); and A–H, B–C and F–G strands (75–80°C). At 80°C, the only identifiable H_N signal was from F105 which indicated that the G–H pair of disulphide-linked strands formed the most heat-resistant feature of the β-Lg structure. The effect of heating to 80°C was shown to be largely reversible by subsequent D/H exchange and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Interestingly, the susceptibility of particular H_Ns to H/D exchange was found to correlate reasonably well with the susceptibility to tryptic hydrolysis.     

Lactobacillus plantarum BC299 can alleviate dextran sulphate sodium-induced colitis by regulating immune response and modulating gut microbiota

It is well known that probiotics are effective in relieving inflammatory bowel disease (IBD). However, their exact mechanism is still unclear. Herein, we evaluated the effect of novel Lactobacillus plantarum BC299 on dextran sodium sulphate (DSS)-induced colitis model mice and investigated the molecular mechanism. We found BC299 could regulate immune response and reduce immunosuppression by enhancing the expression of Th1 T cells in cyclophosphamide-induced immunosuppressive model mice. Meanwhile, DSS-induced colitis model mice were furtherly constructed. The results displayed BC299 can restore bodyweight and relieve colon morphology of colitis mice. Real-time quantitative polymerase chain reaction results demonstrated BC299 can regulate immune response by reducing the expression of TNF-α and IL-1β mRNA and increasing the expression of IL-10 and TGF-β mRNA. Moreover, BC299 administration reshaped and increased the diversity of gut microbiota in IBD mice. Therefore, BC299 can alleviate colitis by regulating immune response and modulating gut microbiota.     

Protective effect of Lactobacillus fermentum CQPC04 on dextran sulfate sodium–induced colitis in mice is associated with modulation of the nuclear factor-κB signaling pathway

Colitis severely affects the quality of life of patients, and lactic acid bacteria have been reported to be able to improve or treat colitis. In this study, we selected a strain of Lactobacillus fermentum (CQPC04) with good resistance in vitro to evaluate its effect on improvement in mice with dextran sulfate sodium (DSS)–induced colitis. We analyzed the effects of L. fermentum CQPC04 on mice with colitis macroscopically via colon length and histopathology. We also used conventional biochemical and ELISA kits, real-time quantitative PCR (RT-qPCR), and Western blotting to analyze microscopically the effects of L. fermentum CQPC04 on related oxidant indices and pro- and anti-inflammatory cytokines in serum and colon tissue of mice. The results indicated that L. fermentum CQPC04 notably increased colon length and ameliorated pathological damage of colon tissue in colitic mice. Serum indices showed that L. fermentum CQPC04 increased the enzyme activity of total superoxide dismutase (T-SOD) and catalase (CAT) and decreased the content of malondialdehyde (MDA) and the activity of myeloperoxidase (MPO). In addition, it inhibited the release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), IFN-γ, IL-1β, IL-6, and IL-12, and increased the release of the anti-inflammatory cytokine IL-10 in serum. The RT-qPCR experiments confirmed that L. fermentum CQPC04 downregulated the expression of pro-inflammatory cytokine nuclear factor-κB-p65 (NF-κBp65), NF-κB inhibitor-α (IκB-α), TNF-α, IFN-γ, IL-1β, IL-6, cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS), and upregulated the expression of IL-10 in colon tissue. Western blot analysis indicated that L. fermentum CQPC04 significantly reduced expression of NF-κBp65, TNF-α, IL-1β, COX-2, and iNOS in mouse colon tissues, and increased expression of IκB-α and superoxide dismutase 2 (SOD2). Thus, L. fermentum CQPC04 could effectively alleviate the symptoms of DSS-induced colitis mice and is a potential probiotic for human experiments.     

Protective effects of galacturonic acid-rich vinegar brewed from Japanese pear in a dextran sodium sulfate-induced acute colitis model

The effects of pear vinegar (PV), which was specially brewed for enhanced galacturonic acid content, on the DSS-induced ulcerative colitis (UC) mouse model were evaluated. PV improved clinical symptoms, colon inflammation, and histological tissue injury in the DSS-induced acute UC mouse model. Moreover, PV suppressed inflammation due to acute UC by suppressing the myeloperoxidase (MPO)-mediated activation of inflammatory cells such as leukocytes and decreasing the serum concentration of IL-6. Our results demonstrated the protective action of PV in the DSS-induced acute UC mouse model. On the other hand, commercial apple vinegar did not show a protective effect in the DSS-induced acute UC mouse model. Our findings indicate that PV may act as a new functional food for inflammatory bowel disease patients.     

Inactivation ofEscherichia coliinoculated in liquid whole egg by high hydrostatic pressure

The resistance ofEscherichia coliin liquid whole egg was studied at several pressures (300, 350, 400 and 450MPa), temperatures (50, 20, 2 and –15°C) and times (5, 5+5, 10, 5+5+5, 15min). The highest reduction was obtained at 50°C (about 7log₈units). At 20 and –15°CE. coliwas more resistant to pressure than at 50 and 2°C. The intermittent treatments were more effective than continuous treatments at lower pressures (350MPa). The destruction increases upon increasing the pressure and the time treatment. Survivor curves were studied at 400MPa for two temperatures (20 and 2°C) and different times (0–60min), obtaining a decimal reduction time of 14.1min at 20°C and 9.5min at 2°X.     

Bilberries and their anthocyanins ameliorate experimental colitis

Bilberries have positive effects in acute and chronic diarrhea. Patients with inflammatory bowel disease (IBD) report on improved symptoms upon ingestion. Bilberries contain approximately 10% of anthocyanins (ACs), which have anti-oxidative, anti-carcinogenic, and anti-inflammatory properties. We investigated whether experimental colitis can be ameliorated by dried bilberries or ACs. Acute and chronic dextrane sodium sulphate (DSS) colitis were induced in Balb/c mice by 2.5% DSS in the drinking water. Mice were fed with dried bilberries or ACs, respectively. Cytokines were determined in supernatants from mesenteric lymph nodes (MLNs) by ELISA and apoptosis was investigated by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assays. Oral administration of bilberries during acute DSS-induced colitis ameliorated disease severity and reduced secretion of IFN-γ and tumor necrosis factor from mesenteric lymph node cells. Dried bilberries also improved chronic DSS-colitis. Ingestion of ACs reduced intestinal inflammation in acute and chronic DSS-colitis with decreased histological scores and cytokine secretion. Both bilberries and ACs prevented inflammation-induced apoptosis in colonic epithelial cells. Taken together, ingestion of dried bilberries had positive effects on various parameters especially in acute DSS-colitis. Oral administration of ACs resulted in an amelioration of acute colitis as well as chronic colitis. These promising results justify a clinical study on their therapeutic effect in inflammatory bowel disease patients.     

鱼腥草多酚对葡聚糖硫酸钠（DSS）诱导小鼠溃疡性结肠炎的改善作用

目的：探究蒿本内酯（ligustilide,Lig）对葡聚糖硫酸钠（dextran sodium sulfate,DSS）诱导溃疡性结肠炎（ulcerative colitis,UC）小鼠的改善作用及机制。方法：将60只雄性SPF级C57BL/6小鼠随机分成6组,分别为空白组、DSS组、阳性药柳氮磺吡啶（sulfasalazine,SASP）组、Lig低、中、高剂量组。DSS组小鼠自由饮用3% DSS水溶液7 d复制UC模型,同时对Lig低、中、高剂量组小鼠灌胃给予药物干预治疗。通过记录小鼠每日体重和结肠长度,检测疾病活动指数（disease activity index,DAI）评分,采用酶联免疫吸附测定试剂盒定量检测血清中TNF-α、IL-6和IL-1β的表达水平,检测结肠中TLR4/NF-κB蛋白表达水平,并结合苏木素-伊红染色实验,探究Lig对小鼠UC的作用及机制。结果：与空白组比较,DSS组小鼠体重显著减轻（P<0.05）,结肠长度显著缩短（P<0.05）,DAI评分显著升高（P<0.05）,肠道出现大量炎症性浸润现象,且结肠组织中TNF-α、IL-6和IL-1β表达水平显著升高（P<0.05）;与DSS组比较,Lig中剂量组和高剂量组小鼠的肠道组织中TNF-α、IL-6和IL-1β的表达以及TLR4/NF-κB信号均被显著抑制（P<0.05）,表明Lig中剂量组和高剂量组小鼠肠道损伤得到显著改善。结论：Lig能有效改善DSS诱导的小鼠UC,其机制可能是抑制NF-κB信号通路的激活。

     

Changes in vitamin content of powder enteral formulas as a consequence of storage

The thiamine, vitamin E (α-, γ- and δ-tocopherol) and vitamin A (all-trans and 13-cis-retinol) contents of four commercial powder enteral formulas (A, B, C and D) have been determined. The vitamin intake provided by the studied formulas was always above the US daily recommendations. Powder enteral formulas A and D were stored at 30 °C for up to 6 months with a water activity of 0.44 (A_w = 0.44), and formula A was also stored under atmospheric conditions for 3, 4 and 6 months. Formulas A and D kept at 30 °C and A_w = 0.44 suffered a gradual loss in vitamin content (from 3% to 4% after 1 month to 58–60% after 6 months). Formula A, stored at 30 °C under atmospheric conditions, underwent a slight reduction in vitamin content after 3 months, similar to that found after 1 month with A_w = 0.44, and from that time onward, this decreased steadily (to 30% after 6 months). The RDA of thiamine, vitamin E and vitamin A for women and men were met only when the powder enteral formulas were stored at 30 °C with A_w = 0.44 up to 1 month and without A_w up to 3 months. These results show that A_w and storage period have a marked effect on the stability of thiamine, vitamin E and A during the storage of powder enteral formulas and should be taken into consideration for the shelf-life of the product.     

小麦面筋蛋白酶解物对小鼠溃疡性结肠炎的影响

以小麦面筋蛋白为原料,采用定向酶解工艺制备富含Gln的小麦面筋蛋白酶解物,并通过葡聚糖硫酸钠(DSS)诱导的小鼠结肠炎模型,探讨酶解物对小鼠溃疡性结肠炎的调节作用。结果表明:小麦面筋蛋白酶解物能缓解DSS诱导的结肠炎小鼠体重降低和炎症临床症状,提高小鼠结肠组织的抗氧化能力,减轻结肠组织炎症因子分泌和炎症细胞的浸润。表明小麦面筋蛋白酶解物能有效缓解溃疡性结肠炎小鼠的肠道炎症。     

Development of postbiotics by whey bioconversion with Enterococcus faecalis M157 KACC81148BP and Lactococcus lactis CAU2013 KACC81152BP for treating periodontal disease and improving gut health

This study developed postbiotics with whey bioconversion product produced by Enterococcus faecalis M157 KACC 81148BP, and mixed whey bioconversion products produced by E. faecalis M157 KACC 81148BP and Lactococcus lactis ssp. lactis CAU2013 KACC 81152BP to alleviate periodontitis (PD) and to improve gut health. The powdered whey bioconversion product (EF) produced by E. faecalis M157 KACC 81148BP, mixed whey bioconversion products (EF+LL) from E. faecalis M157 KACC 81148BP and L. lactis CAU2013 KACC 81152BP, and phosphate-buffered saline (PBS; control) were administered orally to PD-induced rats for 8 wk. Infiltration of inflammatory cells and epithelial proliferation in periodontal tissue were found in control, but the lesions were reduced in PD+EF group (administration of EF to PD-induced rats), and no lesions were observed in PD+EF+LL group (administration of EF+LL to PD-induced rats). The bone loss volumes in PD+EF and PD+EF+LL groups were lower than in control. Cytokine production levels related to inflammation were lower and antioxidative stress markers were higher in PD+EF and PD+EF+LL groups than in control for both periodontal tissue and gut. The ratios of Lactobacillus spp. in gut microbiome of PD+EF and PD+EF+LL groups were higher than in control. These results indicate that the whey bioconversion product produced by E. faecalis M157 KACC 81148BP, and mixed whey bioconversion products produced by E. faecalis M157 KACC 81148BP and L. lactis CAU2013 KACC 81152BP are effective on relieving periodontitis and improving the gut health.     

Temperate and virulent Lactobacillus delbrueckii bacteriophages: Comparison of their thermal and chemical resistance

The aim of this work was to study the efficiency of diverse chemical and thermal treatments usually used in dairy industries to control the number of virulent and temperate Lactobacillus delbrueckii bacteriophages. Two temperate (Cb1/204 and Cb1/342) and three virulent (BYM, YAB and Ib3) phages were studied. The thermal treatments applied were: 63 °C for 30 min (low temperature – long time, LTLT), 72 °C for 15 s (high temperature – short time, HTST), 82 °C for 5 min (milk destined to yogurt elaboration) and 90 °C for 15 min (FIL-IDF). The chemical agents studied were: sodium hypochlorite, ethanol, isopropanol, peracetic acid, biocides A (quaternary ammonium chloride), B (hydrogen peroxide, peracetic acid and peroctanoic acid), C (alkaline chloride foam), D (p-toluensulfonchloroamide, sodium salt) and E (ethoxylated nonylphenol and phosphoric acid). The kinetics of inactivation were drew and T₉₉ (time necessary to eliminate the 99% of phage particles) calculated. Results obtained showed that temperate phages revealed lower resistance than the virulent ones to the treatment temperatures. Biocides A, C, E and peracetic acid showed a notable efficiency to inactivate high concentrations of temperate and virulent L. delbrueckii phages. Biocide B evidenced, in general, a good capacity to eliminate the phage particles. Particularly for this biocide virulent phage Ib3 showed the highest resistance in comparison to the rest of temperate and virulent ones. On the contrary, biocide D and isopropanol presented a very low capacity to inactivate all phages studied. The efficiency of ethanol and hypochlorite was variable depending to the phages considered. These results allow a better knowledge and give useful information to outline more effective treatments to reduce the phage infections in dairy plants.     

Effect of dietary vitamin E on rumen biohydrogenation pathways and milk fat depression in dairy cows fed high-fat diets

Six lactating Holstein cows were assigned to a replicated Latin square design to test the effect of dietary vitamin E on milk fat depression and on the increased production of milk trans-10 C18:1 classically observed when feeding high doses of unsaturated fatty acids with low-fiber diets. Two diets (linseed diet and linseed diet + 12,000 IU of vitamin E/d) were compared during 2 periods of 21 d. The linseed diet presented a forage-to-concentrate ratio of 50:50 and contained extruded linseed (1.86 kg/d) and linseed oil (190 g/d). It was conceived to favor the “trans-11 to trans-10 shift” (low structural value and high level of unsaturated fatty acids). Milk yield and protein content were not affected by the diets. Milk of cows fed the linseed diet presented the typical symptoms of milk fat depression associated with a shift in biohydrogenation pathways: low fat content and high level of trans-10 C18:1. However, the high dose of dietary vitamin E provided significantly increased milk fat content (by 17.93%) and yield (by 15.56%) and decreased trans-10 C18:1 content (by 47.06%). In addition, it managed to significantly increase the daily yields of vaccenic (by 102.56%) and rumenic acids (by 56.67%). However, the sequence of administration of vitamin E influenced its effect, as vitamin E seemed to be more active in limiting the “trans-11 to trans-10 shift” when it was incorporated in the diet simultaneously with the fat. Once the shift had occurred, the subsequent addition of vitamin E was no longer able to completely counteract this process.