Effects of Kluyveromyces marxianus Isolated from Tibetan Mushrooms on the Plasma Lipids , Egg Cholesterol Level , Egg Quality and Intestinal Health of Laying Hens

The effects of the Kluyveromyces marxianus M3 strain, isolated from Tibetan mushrooms, on plasma lipids, egg cholesterol level, egg quality, and intestinal health of laying hens were evaluated. In total, 160 Beijing fatty laying hens (43 weeks old) were divided into four groups and fed a basal diet supplemented with 0%, 0.1%, 0.3%, or 0.5% freeze-dried K. marxianus M3 powder for four weeks. The results showed that yeast supplementation reduced serum total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), and very low density lipoprotein-cholesterol (VLDL-C) levels (p<0.01), and increased serum high density lipoprotein-cholesterol (HDL-C) level (p<0.05). Moreover, regardless of K. marxianus M3 dietary addition level, the cholesterol content of the eggs decreased by more than 26%. When0.3% yeast was supplemented, significant differences were found in the egg weights, shell strength, albumen height, Haugh unit and nutrient content of the eggs (p<0.01). Finally, 0.3% yeast supplementation improved the intestinal flora conditions of the hens by decreasing the Salmonella and Staphylococcus aureus counts (p<0.01) and increasing the Bifidobacterium count (p<0.01). The results in this work demonstrated that yeast culture supplementation to the diets deceased the serum and egg yolk cholesterol, and increased egg quality.


InTRoduCTIon
Yeast is often used as a protein source in animal diets.Yeast and yeast products may also serve as alternatives to antibiotics to promote growth and disease resistance in poultry (Yalçin et al., 2010).In recent years, there has been an increasing number of reports documenting the use of various yeast strains and yeast products, such as inactive dried yeast, yeast culture, whey yeast, selenium yeast, chromium yeast and yeast cell walls, in the diets of laying hens.The inclusion of yeast in layer diets was shown to change egg production (Davis & Anderson, 2002), cholesterol content of the egg yolk (Yousefi & Karkoodi, 2007;Yalçin et al., 2008a), intestinal health of the hens (Yalçin et al., 2010;Lensing et al., 2012), egg quality (Utterback et al., 2005;Yalçin et al., 2008a, b;Yalçin et al., 2009;Yalçin et al., 2010), and egg fat content (Savage et al., 1985;Hosseini, 2011), among others.
The yogurt prepared from Tibetan mushrooms and milk has an extraordinary taste and provides excellent nutrition.Tibetan kefir has a granular structure due to the symbiotic microorganisms present, such as Lactobacillus and yeast (Simova et al., 2002).This complex microbial flora forms a long-term symbiotic relationship and creates a special mucopolysaccharide coat on the surface of the grain.Therefore, Tibetan kefir can protect itself against attack by other microbes and, due toits stable formulation, can be preserved for several years.Hence, Tibetan kefir is an economical and easily preserved material.

Effects of Kluyveromyces marxianus Isolated from Tibetan Mushrooms on the Plasma Lipids, Egg Cholesterol Level, Egg Quality and Intestinal Health of Laying Hens
In recent years, some researchers have found that kefir-fermented milk can decrease the plasma cholesterol levels and promote cancer resistance.Furthermore, it has antioxidant properties and play a role in immune regulation, and may aid the protection against pathogenic bacteria and spoilage organisms, as well as assist in the conservation of the predominant gastrointestinal probiotic flora (Akalin et al., 1997;Abd El-Gawad et al., 2005;Nguyen et al., 2007;Mathara et al., 2008;Zheng et al., 2013).The objective of this study was to evaluate the effects of different doses of Kluyveromyces marxianus M3 yeast isolated from Tibetan mushrooms on plasma and egg yolkcholesterol contents, egg quality, and intestinal flora of the laying hens.

Preparation of yeast strains
Kluyveromyces marxianus M3 was isolated from Tibetan mushrooms and was cultured by a resident of Baicheng, Jilin province, China (Liu et al. 2005).M3 strains (1-2%) were inoculated into 10 mL potato lactose liquid medium and grown at 28°C for 24 hours (h); by this time, the number of living microbes reached 10 7 cfu/mL in the fermentation liquor.An aliquot (3%) of the fermentation liquor was transferred to 1 L of potato lactose liquid medium and fermented for 24 h in a shaker at 32°C.The number of living microbes was counted using a standard plate count (SPC) procedure.The fermentation liquor was then centrifuged for 10 min at 4°C (8000 r/min), and the sediment was saved as the M3 yeast mire.The yeast mire was added to a freeze-drying bottle and then mixed with 1.5% sodium glutamate and 4.5% skim milk powder.This mixture was then frozen at -40ºC for 13 h and freeze-dried at -55ºC for 48 h until completely dried.The yeast powder of K. marxianus M3 was used in the following experiments.

Birds, Diets, and Management
A total of 160 Beijing-You laying hens (Beijing Yufa farm), a commercial white egg strain, with 43 weeks in age, were randomly allocated into a control group and three treatment groups of 40 hens each.Each group was divided into five replicates, comprising of eight hens each.They were housed in cages (30 cm × 50 cm × 50 cm) in a windowed poultry house (four cages per replicate with two birds per cage) under a 16/8 h light/ dark regime.Feed, in the mash form, and water were provided ad libitum during the 4-week experimental period.

Plasma Analysis
At the end of the four-week experiment, three chickens per replicate were randomly selected, and their venous blood was collected when their stomachs were empty.The samples were allowed to stand for 10 min and were then centrifuged at 3500 r/min for 15 min; the sediment was discarded.The TC (total cholesterol), TG (triglyceride), HDL-C (high density lipoprotein-cholesterol), and LDL-C (low density lipoprotein-cholesterol) levels were analyzed using kits (Bio-technology and Science Incorporation) and a fully automatic biochemical analyzer (Hitachi, Japan).The VLDL-C (very low density lipoprotein-cholesterol) was calculated using the following equation: VLDL-C =TC -HDL-C -LDL-C

Assay of egg quality
At the end of the experimental period, four eggs per replicate were randomly collected.Eggs were weighed and hard-cooked by immersion in boiling water for 5 min.Yolks were individually weighed and pooled by blending four yolks per sample, and then stored in a freezer at -20°C.
The yolk cholesterol content was examined using UV spectrophotometry at 550 nm and the o-phthalaldehyde method (Rudel & Morris, 1973).The

Effects of Kluyveromyces marxianus Isolated from Tibetan Mushrooms on the Plasma Lipids, Egg Cholesterol Level, Egg Quality and Intestinal Health of Laying Hens
rate of cholesterol reduction was calculated according to the standard curve of absorbance (y = 0.0919x -0.002, R 2 = 0.9995).The best dose of the M3 live yeast powder was selected for further testing.
The mineral content of the yolk was measured using an inductively coupled plasma atomic emission spectroscopy (ICP-AES) instrument (Spectro Arcos, Kleve, Germany).The total protein concentration was assayed using a Nanjing Jiancheng Bioengineering Institute assay kit (Nanjing, China).The dry matter content was determined using lyophilization, and the crude fat content was examined using the Soxhlet method.The vitamin A and vitamin E content were assayed using high performance liquid chromatography (HPLC), according to the method described by Rubolini et al. (2011).
After four weeks of administration, egg weight (g), yolk weight (g), and eggshell weight (g) of the intermediate yeast dose group and the control group were examined.Eggshell thickness (mm) was measured using a micrometer.Eggshell strength (kg/ cm 2 ) was investigated using an Egg Analyzer (Orka Food Technology, Israel).Albumen height (mm), yolk color, and Haugh unit were automatically analyzed using an EMT-5200 Multi-functional egg quality analyzer (Japan).The Haugh unit (HU) was calculated using the equation: HU =100 log (H − 1.7× W0.37+ 7.6), where H is the observed albumen height (mm), and W is egg weight (g).

Viable
Bifidobacterium, Salmonella, and Staphylococcus aureus present in the excreta were enumerated.This experiment was carried out during the rearing test for the laying hens, and three chickens were randomly selected from replicate of control and 0.3 % K. maxianus M3 supplementation.Every week, 1 g of feces from each hen was collected and stored in weighed, sterilized test tubes.Due to the presence of the anaerobic Bifidobacterium in the feces, the test tubes containing the feces were stored in a Biobag containing a de-oxidant.One gram of excreta was transferred into a test tube containing 9 mL sterile normal saline.The samples were mixed well by vortexing, and serial dilutions from 10 -1 cfu/mL to 10 -8 cfu/mL were prepared.According to the results of the trial test, three dilutions were chosen, and 0.1 mL of this dilution was inoculated into the corresponding medium.Every dilution was run in duplicate.The process of cultivating anaerobic bacteria was completed in 15 min.Bifidobacterium was cultured on MUP-MRS agar plates in Bio-bags at 36ºC±1ºC for 48 h.Salmonella was cultured on bismuth sulfite agar plates at 36ºC ±1ºC for 24 h, and S. aureus was cultured on Baird-Parker agar plates at 36ºC ±1ºC for 18-24 h.Only the typical bacterial colonies were counted, and the total counts for the three types of bacteria were examined.

Date analysis
Statistical analyses were performed using SPSS program (SPSS INC., Chicago, IL, USA) and were expressed as mean ± sd.One-way analysis of variance was used to evaluate the effects of different concentrations of Kluyveromyces marxianus M3 on serum lipids, egg traits, and intestinal flora.Mean differences among groups were compared by the test of Duncan.The level of statistical significance was set at p<0.01.

Plasma lipids
The effects of K. marxianus M3 live yeast supplementation on the serum lipid levels of laying hens are presented in Table 2.After the 4 th week, TC, TG, LDL-C, and VLDL-C levels of the three experimental groups were significantly lower than those of the control group (p<0.01).Conversely, the HDL-C levels were higher in the M3-supplemented hens than in the control hens (p<0.01).The greatest reductions in serumlevels of TC, TG, LDL-C, and VLDL-C were observed in the MD group, of 24.30%, 25.79%, 45.90%, and 36.05%,respectively, whereas their serumHDL-C level increased by 39.06% relative to the control group.In the LD group, serum TC, TG, LDL-C and VLDL-C levels dropped by 20.39%, 21.41%,

Egg Quality
During the four weeks of administration, the cholesterol content of the eggs was examined every week.As shown in Table 3, the cholesterol contents of the eggs from the yeast-fed groups were significantly lower (p<0.01)than those from the control group.Additionally, the cholesterol content of the three yeastfed groups declined gradually during the four-week administration period.At the end of the fourth week, the cholesterol content of the eggs of the MD, LD and HD groups were 229.88 mg/100 g, 313.758 mg/100 g, and 358.17 mg/100 g, respectively, corresponding to 52.8%, 35.28% and 26.46%, respectively, of the cholesterol content of the eggs of the control group.We determined that a highly significant reduction in the cholesterol content of the eggs could be achieved by adding 0.3% M3 freeze-dried powder to the basal diet over a 4-week period, resulting in a reduction of 52.80%.
We further examined the egg quality of the MD group.As shown in Table 4, egg, yolk, and eggshell weights of the MD group were significantly higher (p<0.01)relative to the control group.However, eggshell strength of the eggs laid by yeast-fed hens was lower than that of the control group.Additionally, albumen height and Haugh unit of MD eggs were dramatically higher (p<0.01)than the control group, which suggests better quality of the eggs of the experimental group compared with the control group.However, we did not observe any significant difference in the index production between the yeast-fed and the control hens.
As shown in Table 5, over the course of four weeks, the addition of the 0.3% M3 live yeast preparation to the basal diet not only reduced egg cholesterol content below the international reference value (USDA National Nutrient Database for Standard Reference Release 28), but all the nutrient and chemical values were also significantly higher (p<0.01)than those of the control eggs, especially vitamin A, crude fat, vitamin E, calcium, zinc, selenium, and dry matter.

Intestinal flora
During the experimental period, we examined the changes in the intestinal flora of the hens fed the 0.3% K. marxianus M3-supplemented diet every week.As shown in Table 6, during the first two weeks of the experiment, there were no marked differences in Bifidobacterium, Salmonella, or S. aureus counts between the MD group and the control group.After the third week, the Bifidobacterium count in the MDgroup hens was significantly higher (p<0.01)than that of the control-group hens.After the fourth week, the Bifidobacterium count of the MD group was 9.72 log cfu/g feces, while the control group was only 7.44 log cfu/g feces.Furthermore, the Salmonella and S. aureus counts in the feces of both the MD-group hens and the control hens were similar during the first two weeks of the experiment and then began to dramatically decrease during the third week.After the fourth week, the Salmonella and S. aureus counts in the MD group were 4.47 log cfu/g feces and 5.56 log cfu/g feces, respectively, which were significantly lower (p<0.01)than those of the control group (Table 6).

dISCuSSIon
Eggs are eaten almost daily and contain abundant nutrients, particularly high-quality protein.However, the large amount of cholesterol in the yolk can lead to high blood cholesterol levels in humans, an important factor contributing to atherosclerosis and coronary heart disease (Mohan et al., 1995).Therefore, how to decrease the cholesterol content of eggs is a problem that urgently needs to be solved.Recently, diet manipulation has emerged as a new strategy to improve the nutritional composition of animal products.Supplementing layer feeds with probiotic supplements has been shown to be an efficient and versatile way to lower egg cholesterol level (Mahdavi et al., 2005).
Although the mechanism by which probiotics reduce egg cholesterol content has not yet been studied, we infer that the mechanism is similar to those indicated by some recent studies.It is thought that some lactic acid bacteria may secrete high-activity bile salt hydrolase (BSH) during metabolism, and this secretion could considerably affect cholesterol levels (Pereira & Gibson, 2002;Nguyen et al., 2007).Furthermore, BSH degrades cholates into amino acids and low-solubility free cholate in the enterohepatic circulation.The free cholate and cholesterol combine into sediment composites, which are then expelled along with the feces.Subsequently, the free cholate in the liver enterohepatic circulation decreases, thus increasing the biosynthesis of cholic acid; this increase in cholic-acid synthesis accelerates the catabolism of cholesterol, which is the precursor ofcholic acid.Thus, total cholesterol plasma level decreases (Mann & Spoerry, 1974;Liong, 2005;Begley et al., 2006).
According to our results, supplementing the basal diet of laying hens with K. marxianus M3 live yeast clearly decreased the serum TC, TG, LDL-C, VLDL-C levels and increased the serum HDL-C level (Table 2).VLDL is the main storage area or carrier of plasma cholesterol, which combines both cholesterol and the yolk cholesterol.Therefore, a lower VLDL-C value means there is less cholesterol in the egg.Furthermore, the cholesterol content of the egg yolks of the group fed the K. marxianus M3-supplemented diet was significantly lower than that of the control group (p<0.01).After the fourth week, the egg cholesterol content of the LD, MD and HD groups was reduced by 35.58%, 52.80% and 26.46%, respectively (Table 3).Our results indicate that there was a relationship between the synthesis and reduction of egg cholesterol and the metabolism of cholesterol in the plasma.Recently, considerable attention has been given to the potential of probiotics in altering lipid metabolism.This interest stems from the growing evidence that probiotics reduce cholesterol concentration in the egg yolk (Abdulrahim et al. 1996;Haddadin et al., 1996;Panda et al., 2003;Kalavathy et al., 2009) and in the serum (Kurtoglu et al., 2004;Alkhalf et al., 2010).
In this study, we further demonstrated that feeding laying hens a diet containing K. marxianus M3 live yeast increased yolk weight, eggshell weight, albumen height, and Haugh unit (Table 4); however, we did not observe any significant differences in egg production between the yeast-fed and the control hens.Moreover, the egg concentrations of vitamin A, crude fat, vitamin E, calcium, zinc, selenium, and dry matter also dramatically improved in the eggs of yeastfed hens (Table 5).Similar studies reported a significant improvement of egg production in hens fed a mixed culture of probiotics, such as Lactobacillus acidophilus and Lactobacillus casei (Tortuero & Fernandez, 1995), L. acidophilus alone (Haddadin et al., 1996), Pediococcus acidilactici (Mikulski et al., 2012), Bacillus subtilis (Xu et al. 2006;Abdelqader et al. 2013), Propionibacterium jensenii (Luo et al., 2010), and Saccharomyces cerevisiae (Yalçin et al., 2010).Significant improvements in egg weight and eggshell quality were also obtained in hens fed diets supplemented with a mixture of Lactobacillus cultures (Davis & Anderson, 2002;Kalavathy et al., 2009).
The intestinal microflora plays a major protective role in maintaining the integrity of the intestinal mucosa.The addition of beneficial bacteria to a diet can recover the intestinal integrity, thereby increasing nutrient bioavailability and absorption (Burel & Valat, 2009;Deng et al., 2012).Probiotics also improved gut health, which directly improved the birds' health and performance (Burel & Valat, 2009).In this study, we examined the presence of three typical intestinal flora components in the feces of laying hens.Our results indicated that throughout the entire experimental period, the numbers of Bifidobacterium present in the feces of the control group steadily grew; however, the Bifidobacterium counts in the MD groups were higher than those in the control group after the third week (Table 6).Moreover, the Salmonella and S. aureus counts in MD group decreased compared to the control group (Table 6).Therefore, we concluded the M3 live yeast modulates the intestinal flora and protects the intestinal tract of laying hens.

dISCloSuRe
There is no conflict of interest with other works.
This research was funded by the Comprehensive Reforming Project to promote talents training of BUA, Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions (CIT&TCD20140315) and the Key Construction Discipline Program of Beijing Municipal Commission of Education (PXM2014-014207-0000029).

Table 1 -
Ingredients and chemical composition of the basal diet.

Table 2 -
Effect of the M3 live yeast preparation on the serum lipid levels of laying hens ( x±s, n=15) a-b: Means within a column followed by the same superscript are not significantly different (p<0.01).

of Kluyveromyces marxianus Isolated from Tibetan Mushrooms on the Plasma Lipids, Egg Cholesterol Level, Egg Quality and Intestinal Health of Laying Hens 39
.34% and 30.04%, respectively, whereas the HDL-C level increased by 32.81% compared with the control group.Moreover, the HD group presented the least reductions of TC, TG, LDL-C and VLDL-C serum levels, which decreased only by 13.97%, 14.95%, 36.07%and 18.03%, respectively, and the HDL-C level increased by 21.88% relative to the control group.Therefore, we concluded that supplementing the basal diet of the laying hens with K. marxianus M3 live yeast clearly decreased TC, TG, LDL-C, VLDL-C serum levels and increased HDL-C serum level.

Table 5 -
Effect of the M3 live yeast preparation powder on the nutrient and chemical indices of the eggs ( x ±s,

Table 3 -
Effect of the K. marxianus M3 freeze-dried powder on the cholesterol content of the eggs ( x ±s, n=20).
a-d: Means within a column followed by the same superscript are not significantly different (p<0.01),"D" represents the percent decrease in the cholesterol content of the eggs from the former week in each group.

Table 4 -
Effect of dietary K. marxianus M3 live yeast preparation powder on egg quality ( x ±s, n=20)

Table 6 -
Effects of diet probiotic on excreta microflora in laying hens (log10 cfu/g feces, x±s, n=15) a-c: Means within a row with no common superscript differ significantly (p<0.01)Effects