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).
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