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Agriculture and Food Science Vol. 1 (1), pp. 006 - 010 August, 2012. Available online http://www.heraldjournals.org/hjafsr/archive.htm Copyright (c) 2012 Herald International Research Journals. Effects of fermented Camilla sinensis, Fuzhuan tea, on
egg cholesterol and production performance in laying
‡Xiaojiang Xu1,2, ‡Yabei Hu1, *Wenjun Xiao1,2, Jian’an Huang1,2, Xi He1, Jin Wu1, Elizabeth P.
Ryan3, and *Tiffany L. Weir4
1Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hunan Agricultural University, Changsha, China,2National Research Center of Engineering and Technology for Utilization of Functional Ingredients from Botanicals, Changsha, Hunan, China, 410128; 3 Department of Clinical Science and 4 Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, CO 80523. Fuzhuan tea, which is a Chinese dark tea produced by fermentation with the non-pathogenic fungus
Eurotium cristatum, has reportedly demonstrated lipid lowering effects in rat models. These published
studies suggest that fuzhuan tea may be a suitable feed additive for laying hens to reduce egg
cholesterol levels. Therefore, a 5 week feeding study was conducted to determine the effects of tea-
supplementation on egg cholesterol and production performance in laying hens. A total of 136 laying
hens were randomized into three diet groups and fed ad libitum a basal diet (control), or diets
supplemented with milled fuzhuan tea or its water-soluble extracts(treatment groups). Tea
supplemented hens demonstrated significant improvement in laying performance with increased egg
production of 36% (milled tea) and 24% (tea extracts) compared to control animals. Both treatments also
resulted in decreases in serum total cholesterol (TC) and egg yolk cholesterol. No significant
differences between groups were observed with respect to egg weight, yolk ratio or other serum lipid
parameters. In conclusion, supplementation of chicken feed with fuzhuan tea may be a safe and
effective means of improving feed efficiency and production in layer hen operations while resulting in
eggs with a lower cholesterol content.
Key words: layer hen, Fuzhuan tea, Camellia sinensis, HPLC, total cholesterol, egg production, feed efficiency

INTRODUCTION

Eggs are one of the most widely consumed animal food
investigations suggest that diets high in saturated fats products and are generally considered to be an important rather than high in cholesterol have a greater impact on source of unsaturated fats, essential amino acids, folate and other B vitamins. There is also some evidence to CVD risk, and that about 75% of the population show little suggest that consumption of eggs can decrease blood or no increase in plasma cholesterol levels when glycemic index (Pelletier et al., 1996) and raise high- challenged with a high cholesterol diet (Fernandez, density lipoprotein cholesterol (HDL) levels compared to 2010). Despite new findings, the American Heart carbohydrate rich diets (Packard et al., 1983; Schnohr et Association’s Nutrition Committee still recommends al., 1994). A 20 ounce egg contains between 186-213 mg of cholesterol, and some studies have linked high limiting dietary cholesterol intake to 300 mg per day or dietary cholesterol to increased risk of cardiovascular 200 mg per day for people with heart disease or diabetes disease (CVD) (Krauss et al., 1996). However, recent (Lichtenstein et al., 2006). As egg consumption has tripled worldwide over the past 40 years, a new market for “designer” eggs with value added traits such as increased omega-3 fatty acids or decreased cholesterol has emerged (Hernandez et al., 2009). To meet this Table 1. Chemical major components of Fuzhuan tea.
Table adapted from data on Fuzhuan tea chemical
composition in previously published work (Wu et al., 2010)
Fuzhuan Tea Components
Amount (mg/g)
Catechins
Total Polyphenols
Polysaccharides
Amino Acids
Organic Acids
demand, new approaches to alter the nutritive properties fuzhuan tea and its water-soluble extracts as feed of eggs are being developed, including genetic selection additives for laying hens to determine their effects on of laying hens and exploring feed additives that convey positive egg traits (Wang and Tong, 2002). Tea (Camellia sinensis L.) is the second most consumed beverage world-wide and contains bioactive MATERIALS AND METHODS
compounds such as polyphenols, caffeine, theanines, and vitamins. Numerous health benefits have been Raw Material and Sampling. Fuzhuan tea: Special attributed to these phytochemicals including anti-oxidant package "Golden Xiangyi" Brick Tea produced in 2007 and anti-cancer properties as well as the regulation of was obtained from Yiyang Tea Manufacturer in blood lipid metabolism (Zhao, 2003; Chen, 2009; Yung et Changsha, Hunan, China. Milled samples were produced al., 2008; Alshatwi et al., 2010). Fuzhuan tea (aka Fu by pulling apart compressed tea bricks by hand and Brick tea, PHatea), a Chinese tea whose leaves undergo milling with a comminutor. Water-soluble extracts were a unique process of fermentation with the fungus produced by brewing loose leaf fuzhuan tea with sterile Eurotium cristatum (Raper and Fennell) Malloch and distilled water for 45 minutes at 90°C at a ratio of 1:8 Cain, has distinct phytochemical profiles compared to (w/v) followed by a second extraction of 1:7 (w/v). The other teas (Wu et al., 2010; Luo et al., 2012; Fu et al., two fuzhuan tea extracts were combined and then 2008). Several studies suggest that consumption of fuzhuan tea has significant human health effects on phytochemical analysis of the fuzhuan tea based on data metabolic regulation of blood lipids (Xiao, 2007; Fu et al., obtained from previously published reports (Wu et al., 2011), and thus could potentially lower egg cholesterol levels when used as an additive in chicken feed. Animals and Diets. One hundred thirty six 22-week old Therefore, the objective of this study was to test milled Roman brown laying hens with a mean body weight of Table 2. Ingredient and nutrient composition of the basal diet fed to laying hens
Metabolizable energy (kcal/kg)= 2,507 * provides (mg/kg diet): retinol 2.4, cholecalciferol 0.075, DL-α tocopherol acetate 20, menadione 2, thiamin 1.5, riboflavin 6, pyridoxol 3.5, cobalamin 0.01, niacin 25, panthotenic acid 8, folic acid 1, d-biotin 0.03, ascorbic acid 30, choline chloride 600; Mn 80, Fe 60, Zn 60, Cu 5, I 1, Co 0.2, Se 0.15 1500-1725 grams were individually housed in wire cages the mean egg weight for each group was calculated. Six at Hunan Animal Husbandry Veterinarian’s Research eggs were chosen at random from each treatment group Institute under approved animal handling protocols. All on a weekly basis and used to determine yolk size. Each the animals were fed a pelleted basal dry diet consisting egg was weighed individually and then the yolks were primarily of corn, soybean meal, and rice bran twice per separated from the albumen and weighed. The yolk ratio day (Table 2). Hens were randomly allocated into 3 was calculated as grams per yolk: grams per egg. treatment groups: 1) basal diet amended with milled Yolk cholesterol. Eight eggs were collected weekly
fuzhuan tea at a concentration of 11.2 g /kg of feed from each group for determination of cholesterol levels in (n=48), 2) basal diet amended with 5.7 g of water-soluble egg yolks. Cholesterol in the yolk was extracted and tea extracts per kg of feed (n=40), or 3) a control group measured by High Pressure Liquid Chromatography fed unamended basal diet (n=48). Tea concentrations (HPLC) according to published protocols (Wang and used in this study were determined by preliminary Tong, 2002; Wang, 2002). Eggs were weighed and then experiments and previously published studies in other the egg whites were removed and the egg yolk/total egg animal models (Alshatwi et al., 2010). The experiment weight ratio was calculated. Egg yolk suspensions were was conducted over a 5-week period with all the laying prepared by dissolving 6 g of egg yolk in distilled water in hens kept under uniform management conditions 10 mL volumetric flask. One milliliter of egg yolk throughout the experimental period. Temperature of 25- suspension was added into a 5 mL centrifuge tube and 34°C and a photoperiod of 16.5 h light were maintained. 110 µl ethanol containing 210 mol/L KOH was added and Production traits. Egg production was recorded daily mixed thoroughly. The mixture was saponified by for each individual hen and was calculated every week on incubating at 55⁰C for 30 min, shaking every 5 min. The a bird/day basis. Eggs from each group were collected mixture was cooled and 210 µl n-hexane: isopropanol daily and stored at 4⁰C until further analysis. The eggs (4:1) solution was added and mixed prior to centrifugation produced each week were weighed and the average daily at 3000 rpm for 3 min. The supernatant was removed and weight of the eggs produced was calculated as the extraction procedure was repeated 4 times per grams/hen/day. A total of 1162 eggs were analyzed from sample, combined and allowed to air dry. Concentrated hens in the milled tea group, while 813 eggs were extracts were resuspended in methanol and passed analyzed in the tea extract group, and 512 eggs from through a 0.45uM filter prior to HPLC analysis. Ten hens fed control diets. Feed intake was recorded weekly microliter injections of sample were analyzed on a and calculated as grams per hen per day. The value of Shimadzu SCL-10ATVP system equipped with a feed efficiency was calculated as a ratio of grams of feed: Shimadzu LC-10ATV pump, SPD-M20A diode array detector, and LC-solution data system (Shimadzu Egg Traits. At the end of the experimental period, total Corporation, Japan) using an Ultimate C-18 reverse- egg weight for each of the three groups was obtained and phase column with pore size of 4.6x150mm (Welch Table 3. Production performance of laying hens in three dietary groups.
Egg weight Daily egg weight Feed intake Feed Conversion Yolk ratio (%) *(± standard deviation) a: denotes a significant difference from control values at P＜0.05 Table 4. Effects of Fuzhuan tea on laying hen serum lipid parameters
triglyceride
cholesterol HDL/TC HDL/LDL
Milled Tea
Tea Extract 37.5 ±16.6 57.2 ±31.3 513.3 ±77.0
TC=total cholesterol, TG=Triglycerides, HDL=high density lipoprotein, LDL=low density lipoprotein Materials, Shanghai, China). The column temperature production performance and egg traits are shown in Table was maintained at 38⁰C and a mobile phase of 100% 3. Compared with the control group, the inclusion of methanol was delivered isocratically at a flow rate of 1.0 milled fuzhuan tea in the diet of laying hens resulted in mL/min. All solvents and chemicals used were HPLC significant increases in egg production (36.8%, p<0.01), grade and a commercially purchased cholesterol average daily egg weight (31.6%, p<0.01) and average standard (purity=99%, RMHot, Beijing, China), showing a daily feed intake (27.3%, p<0.01). Feed conversion ratio retention time of 9.3 min under the described conditions, in these animals was also increased by 8.3% (p<0.01). A was used to generate a standard curve used to quantify linear regression of feed intake and egg production showed a significant positive correlation between feed Serum parameters. Blood lipid parameters were intake and egg production (R2 =0.683, P<0.05). Less obtained from blood samples collected at slaughter from pronounced effects were reported for inclusion of fuzhuan six randomly selected laying hens from each group. tea extracts compared to the milled tea; however, Collected blood was centrifuged to separate out the significant benefits in production and efficiency were serum for determination of blood lipid levels. Total observed. Hens fed fuzhuan tea extracts showed cholesterol (TC), triglycerides (TG), high density increased production, higher average daily egg weight lipoprotein (HDL-C) and low density lipoprotein (LDL-C) and average daily feed intake by 24.1% (p<0.05), 19.1% were assayed using commercial kits (Jiancheng (p<0.05) and 23.1% (p<0.01) respectively. No significant Biochemical Company, Nanjing, China) and measured on difference in the values of egg weights and yolk ratio was a MINDRAY Auto Chemistry Analyzer, BS-200 (Mindray, observed among the groups, suggesting that basic egg traits were not affected by the dietary treatments. Statistical analysis. All statistical analyses were done Levels of total serum cholesterol (TC), triglycerides using SPSS 17.0 program (IBM China Company, Ltd., (TG) and high and low density lipoproteins (HDL-C, LDL- Beijing, China). The difference between means of the C) in laying hens fed basal diet or milled fuzhuan tea and treatment and control groups was tested for significance its water soluble extracts were measured at the by Student’s t-test limit set at p<0.05 (significant) or completion of the trial and are shown in Table 4. No significant differences were observed among the three groups in any of the lipid parameters measured. Despite a lack of change in serum lipid levels of the laying hens, there were significant decreases in yolk total The effects of milled fuzhuan tea and its extracts on cholesterol as a result of milled fuzhuan tea and its water soluble extracts. The mean total cholesterol in yolks from strong relationship between serum total cholesterol and the control group was 190.7 ± 26.6 mg/ dL, while yolk TC egg cholesterol is lacking (Shivaprasad and Jaap, 1977; for milled fuzhuan tea and its water-soluble extracts were Wang and Pan, 2003), while others suggest that a 178.0 ± 23.9 mg/dL and 170.3 ± 29.0 mg/dL-1, reduction in serum TC results in reduced yolk cholesterol respectively; which is well below the average egg (Kurtoglu and Nizamlioglu, 2004; Azeke and Ekpo, 2008; cholesterol of 186-213 mg/dL average (Krauss et al., Khan et al., 2007). In the present study, there were no 1996). Thus, supplementation with milled fuzhuan tea significant changes in serum lipid levels, but yolk lowered yolk TC by 6.7% (p<0.05), and the water soluble cholesterol was lower in the tea fed animals, in extracts decreased TC by 10.7% (p<0.05) compared to concurrence with published reports that suggest that the serum and egg cholesterol levels are unrelated. An inverse ratio between serum HDL/LDL ratio and total cholesterol in eggs has also been noted (Wang and Pan, DISCUSSION
2003) and may be an important parameter to target for Modifying egg composition by supplementing the diet of relationship was not noted in our study (Table 3). laying hens has been used to enhance levels of omega-3 However, conflicting reports regarding the relationship fatty acids (Kirubakaran et al., 2011); and similar attempts between serum and egg lipids suggest that more have been made in producing egg yolks with lower research is needed and that genetic differences in the cholesterol (Wang and Pan, 2003). We observed a breeds of laying hens, which affect egg cholesterol levels, significant reduction in egg total cholesterol levels by incorporating fuzhuan tea and its water-soluble extracts Antioxidant compounds were previously shown to into the diets of laying hens. Supplementation of basal reduce the content of cholesterol in egg powders (Du and chicken feed with milled tea and its extracts also Ahn, 2000; Khan et al., 2007), and tea polyphenols and improved egg production, and increased feed intake and other compounds have long been utilized as natural a higher feed conversion ratio, but did not demonstrate antioxidants (Zhao, 2003). The action of tea polyphenols any negative effects on egg weight and yolk ratio, on lipid metabolism in laying hens and reducing egg suggesting that supplementing chicken feed with this tea cholesterol content has also been observed and reported may be a feasible approach to reduce overall costs for (Wang and Tong, 2002; Lou et al., 2004; Khan et al., egg production while resulting in a value-added final 2007). While it is possible that tea polyphenols contribute to the results seen in this study, these compounds are Fuzhuan tea has a unique chemical profile compared reduced in fuzhuan tea compared with other teas to other teas because it undergoes a microbial suggesting that unique compounds resulting from the fermentation. When compared with unfermented teas, fungal fermentation might be involved in observed anti- fuzhuan tea is lower in polyphenols (catechins), caffeine, lipidemic effects, particularly organic acids with anti- and amino acids but has an increased level of organic oxidant activity (Purvis, 2001; Nath et al., 1995), that acids (Fu et al., 2008; Wu et al., 2010). Organic acids occur in higher levels in this tea and are highly water reduce intestinal pH to inhibit the growth of microbial soluble. In fact, the solubility of these organic acids may pathogens, improve the absorption and conversion of account, in part, for the lower egg cholesterol reported for nutrients in the body, and improve overall gastric function (Park et al., 2009; Roth and Kirchgessner, 1998). This The results of the present study suggest that may account for the increased feed intake observed in supplementing the diet of laying hens with fuzhuan tea this study in the tea supplemented animals. The could significantly improve egg production and feed increased food intake was positively correlated with efficiency and reduce egg cholesterol. Both hen serum increased egg production and improved feed conversion. and egg cholesterol can vary dramatically with breed and Cholesterol is present in the yolk of eggs as very low age of laying hen and future studies directed toward density lipoprotein cholesterol (VLDL-C). Unlike in identifying the optimal dose for fuzhuan tea-mediated mammals, where TC and TG are transformed into LDL-C reduction of egg cholesterol taking these additional and HDL-C, (Chen and Zhang, 2003) in chickens, egg variables into consideration are needed. Fuzhuan tea cholesterol is biosynthesized in the liver (Andrews et al., also has reported anti-microbial properties (Liu et al., 1968), secreted into the plasma as VLDL-C (Burley et al., 2010), and it would be worth exploring affects of feed 1984), and transported to the ovaries (Nimpf and supplementation on reduction of Salmonella and other Schneider, 1991). Some reports have suggested that a potential pathogens that decrease the health of hens or that enter the human food supply in eggs. Lichtenstein AH, Appel LJ, Brands M, Carnethon M, Daniels S, Franch HA, Franklin B, Kris-Etherton P, Harris WS, Howard B, Karanja N, Lefevre M, Rudel L, Sacks F, Van HL, Winston M, Wylie-Rosett J ACKNOWLEDGEMENTS
(2006). Diet and Lifestyle Recommendations Revision 2006. Circulation 114(1): 82-96. Liu ZY, Xu AQ, Li ZJ, Wang YL (2010). Research progress on Eurotium Authors express their sincere gratitude and appreciation cristatum and its metabolites in Fuzhuan tea. Tea Communication 37: to the Ministry of Science and Technology of China (2012 APEC funds and 2011BAD10B00), the Denver Diabetes Lou HX, Lin Z, Wang YM, Lu FZ, Tang JF, Ying JF, Yang ZM (2004). Foundation, and the Colorado Agricultural Experiment Effect of dietary tea polyphenols on performances, lipid metabolism and egg quality of laying hens. J. Tea Science 2: 135-140. Station for providing financial support and to the YiYang Luo ZM, Ling TJ, Li LX, Zhang ZZ, Zhu HT, Zhang YJ, Wan XC (2012). tea factory for kindly providing test material. A New Norisoprenoid and Other Compounds from Fuzhuan Brick Nath KA, Ngo EO, Hebbel RP, Croatt AJ, Zhou B, Nutter LM (1995). REFERENCES
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