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Effects of Milk Products Fermented by Bifidobacterium longum on Blood Lipids in Rats and Healthy Adult Male Volunteers

J. Z. Xiao^*, S. Kondo^*, N. Takahashi^*, K. Miyaji^*, K. Oshida, A. Hiramatsu^*, K. Iwatsuki^*, S. Kokubo^* and A. Hosono

^* Food Research and Development Laboratory,
Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd., Zama 228-8583, Japan;
Department of Agriculture, Shinshu University, Nagano-Minamiminowa 399-4598, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The effects of milk products fermented by Bifidobacterium longum strain BL1, a probiotic strain, on blood lipids in rats and humans were studied. Rats were fed a cholesterol-enriched experimental diet, supplemented with lyophilized powders of 1) acid milk (control), 2) milk fermented with a mixed culture of ordinary yogurt starters composed of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (SL), and 3) bifidobacterium milk fermented with the probiotic B. longum strain BL1, respectively. The bifidobacterium milk feeding brought about significant lowering of the serum concentrations of total cholesterol, low-density lipoprotein cholesterol, and triglycerides, in comparison with the control, while no change in high-density lipoprotein cholesterol concentration was observed. On the other hand, supplementation with SL milk resulted in only slight, nonsignificant decreases in serum lipid concentrations in comparison with the control. In the human study, 32 subjects with serum total cholesterol ranging from 220 to 280 mg/dl were randomly assigned to two treatments: 1) intake of a low-fat drinking yogurt prepared with ordinary yogurt starters composed of S. thermophilus and L. delbrueckii subsp. bulgaricus (P-group) and 2) intake of a low-fat drinking yogurt prepared with the two ordinary yogurt starters plus B. longum strain BL1 (B-group). After intake for 4 wk at 3 x 100 ml/day, reduction of serum total cholesterol was observed in approximately half of the B-group subjects; a particularly significant decrease in serum total cholesterol was found among subjects with moderate hypercholesterolemia (serum total cholesterol > 240 mg/dl). However, the serum lipid concentrations in the P-group subjects were almost stable during the experimental periods. The present results indicate the potential of the probiotic B. longum strain BL1 in serum lipid improvement.

Key Words: Bifidobacterium • blood lipid • fermented milk

Abbreviation key: HDL = high density lipoprotein, LDL = low density lipoprotein, IHD = ischemic heart disease, SL = mixed culture of , S. thermophilus and L. delbrueckii subsp. bulgaricus

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
A high level of serum total cholesterol is generally considered to be a risk factor for coronary heart disease and atherosclerosis in the USA, Europe, and Japan (Anderson et al., 1987; Law et al., 1994; Ross, 1993; Yamamoto et al., 1999). Reducing the level causes decreases in incidence and mortality of ischemic heart disease (IHD) and atherosclerosis (Frick et al., 1987; Mabuchi, 1999). Much attention has thus been drawn to different dietary ways of reducing the serum total cholesterol level.

Mann and Spoerry (1974) first reported a hypocholesterolemic effect in Maasai tribesmen of milk fermented by wild type starters. Thereafter, many studies have been performed in experimental animals and humans to elucidate the effect of fermented dairy products on serum cholesterol, especially with selected strains of lactic acid bacteria (see review of St-Onge et al., 2000). The mechanisms of the hypocholesterolemic activity of lactic acid bacteria have been proposed to involve inhibition of exogenous cholesterol absorption from the small intestine by the binding of cholesterol and bile acids with the bacterial cells, assimilation of cholesterol, as well as suppressing bile acid resorption by deconjugation as a function of the bacterial bile salt hydrolase activity (Gilliland et al., 1985; Danielson et al., 1989; Salminen and von Wright, 1993; De Smet et al., 1998).

To guarantee a probiotic effect after administration, microorganisms should survive passage through the gastric juice and small intestine (Gilliland and Walker, 1990). Bifidobacterium longum strain BL1 was found to have a high tolerance for artificial gastric juice and bile (see Results). This strain was found to possess a strong bile salt hydrolase activity (unpublished data). It is thus suspected that strain BL1 would survive passage through the gastrointestinal tract and would function in vivo in cholesterol removal.

Based on the above, the present study was conducted to demonstrate the effect of milk fermented by B. longum strain BL1 on blood lipids in rats and humans.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Source and Maintenance of Cultures
B. longum strain BL1 (MCC 1083) and yogurt starters of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were stock cultures of Morinaga Milk Industry Co., Ltd., Kanagawa, Japan. The culture of B. longum strain BL1 was maintained by subculture (10% inoculation) in 11% reconstituted skim milk containing 0.2% yeast extract at 37°C for 5 to 6 h. The mixed culture of S. thermophilus and L. delbrueckii subsp. bulgaricus (SL) was maintained by subculture (3% inoculation) in 10% reconstituted skim milk at 37° for 5 h. Subcultures were stored at 4°C between transfers. Freshly prepared seed cultures were used for production of yogurts.

Measurement of Gastric Juice and Bile Tolerance
The culture of B. longum strain BL1 was prepared as described above. To 100 ml artificial gastric juice (containing 0.2% pepsin, 0.35% sodium chloride, adjusted to pH 3.0 by HCl), 0.01 ml of culture was added, and the mixture was mixed and incubated at 37°C for 1.5 to 2.0 h. The pH of the mixture was 3.04, and incubation was stopped by adding an equal volume of cooled 10% pasteurized reconstituted skim milk. As a control, 0.01 ml of culture was incubated in 100 ml of 0.86% sodium chloride solution (pH 6.5) at 37°C for 2 h. Viable counts were determined immediately after incubation on Reinforced Clostridial Agar (Oxoid, England) under anaerobic conditions at 37°C for 72 h. The rate of gastric juice tolerance was calculated as follows:

For the bile tolerance test, 0.01 ml of culture was added to 100 ml of 10% pasteurized reconstituted skim milk containing 0 to 1.6% of oxgall (Sigma Chemical Co., St. Louis, MO), and the mixture was incubated at 37°C for either 5 or 16 h. The control was a culture without incubation. Viable counts were determined as described above, and the rate of bile tolerance was calculated as follows:

Animal Experiment
For the production of SL milk, bifidobacterium milk, and control acid milk, skim milk base (containing 11% skim milk powder, 1% glucose, and 0.2% yeast extract) was pasteurized at 90°C for 10 min prior to fermentation, cooled to 40°C, and then divided into three parts. One portion was inoculated with a 1% (w/w) mixed seed culture of ordinary lactic acid bacterial starters composed of S. thermophilus and L. delbrueckii subsp. bulgaricus for SL milk, and the second portion was inoculated with a 1% (w/w) seed culture of B. longum strain BL1 for bifidobacterium milk, respectively. Each of the inoculated mixes was incubated at 37°C until it reached pH 4.5 and then cooled to 4°C. The products were lyophilized for experimental diets. Prior to lyophilization, the fermented milks were partially neutralized by mixing with equal volumes of pasteurized 20% skim milk base to improve bacterial survival when frozen. Preparation of lyophilized powder was carried out three times each for SL milk and bifidobacterium milk. Total viable cfu were 3.8 ± 0.9 x 10⁸/g and 4.1 ± 0.7 x 10⁸/g of lyophilized powder for SL milk and bifidobacterium milk, respectively. Lactic acid was added to the third portion of milk to pH 4.5 for control acid milk, which was neutralized and lyophilized in the same manner as the fermented milks.

Twenty-one rats (Male Sprague-Dawley), that were 4 wk old, were purchased from Japan SLC Co., Ltd., Shizuoka, Japan. After being given a standard diet (F2, Oriental Yeast Co., Ltd., Tokyo, Japan) for 1 wk, they were divided into three groups (seven each) balanced according to BW and randomized to experimental diets that were supplemented with lyophilized powders of SL milk, bifidobacterium milk, or control acid milk. They were housed in an air-conditioned room at 25 ± 2°C with 55 ± 5% humidity on a 12 h light-dark cycle (light from 0800 to 2000 h and dark from 2000 to 0800 h). Water was freely available, and the experimental diets were given at approximately 20 g/100 g BW per day.

The composition of the experimental diet was based on AIN-93M, with protein content of 14% (Table 1). The lyophilized powder was added to the diet at the expense of sucrose and starch. Since large numbers of the lyophilized cells described above were found to die after being combined with the diet, presumably due to the moisture of the diet, the major components (casein, corn starch, -corn starch, sucrose, cellulose, and mineral mixture) were oven-dried at 60°C overnight prior to diet preparation. Diet materials, except lyophilized powders, were mechanically mixed, subdivided, and packaged in sealed aluminum bags and stored in a refrigerator (4°C) until use. The lyophilized powders were mixed with the base diet just before being given to the animals, and freshly prepared diets were replaced daily at 1600 to 1700 h to prevent loss of bacterial survival during storage and feeding. Counts of viable lactic acid bacteria in the diets were between 10⁷ to 10⁸ cfu/g in the freshly prepared diets but decreased to approximately half and 1/10 of the original counts after administration for SL milk and bifidobacterium milk, respectively (Table 1).

Fecal total bile acids were determined by the methods of Hashimoto et al. (1999) with some modification. Milled freeze-dried feces (0.05 g) were extracted twice with 3.5 ml ethanol at 80°C for 1 h. After two extractions, the ethanol was evaporated under N₂ gas at 50°C, and the residue was dissolved in 2.5 ml ethanol. The amounts of total bile acids were analyzed with a commercial test kit (Total Bile Acid Test Wako, Wako Pure Chemical Industries, Ltd., with a detection sensitivity level of 10 to 50 µmol/l of bile acids).

Human Study
The bifidobacterium and placebo drinking yogurts were produced in the Research and Information Center of Morinaga Milk Industry Co., Ltd. The bifidobacterium yogurt was fermented with a starter of B. longum strain BL1 in the presence of ordinary lactic acid bacterial starters composed of S. thermophilus and L. delbrueckii subsp. bulgaricus. The placebo yogurt was fermented with the same ordinary lactic acid bacterial starters employed in preparation of the bifidobacterium yogurt. The fermented milks were then homogenized aseptically at 15 MPa to make drinking yogurts. The properties and cfu of the products are shown in Table 2. The bifidobacterium yogurt contained more than 10⁸ cfu of B. longum strain BL1 when tested on both d 0 and d 9 after production. One hundred grams of each of the bifidobacterium and placebo yogurts had an energy content of approximately 50 kcal and contained 3.0 g of protein, 8.2 g of carbohydrate, and 0.6 g of milk fat. Products were prepared weekly and distributed to the participating subjects in plastic bottles containing 100 ml, which were kept in a refrigerator at 4°C.

Since the present study was a pilot study for future long-term study, it was designed simply as a short-term, single-blind, parallel group study. Starting 1 wk after the pre-intake blood sampling, subjects were instructed to consume 3 x 100 ml of either bifidobacterium or placebo yogurt daily as a part of their habitual meals (breakfast, lunch, and dinner) for 4 wk. During the intake period, subjects had to fill in a questionnaire on compliance, intake of medicine, alcohol consumption, and BW (once a week, wearing indoor clothing without shoes). Blood samples for the measurement of glucose and serum lipids were taken after fasting overnight before breakfast during wk 4.

Blood samples were drawn from an antecubital vein. The analysis of the collected blood samples was performed at BML Co. Ltd., Tokyo, Japan. Total cholesterol, HDL-cholesterol, and triglycerides were measured with a 7450 Automatic Analyzer (Hitachi Co., Ltd., Tokyo, Japan). Blood glucose was measured with a JCA-BM12 Automatic Analyzer (JEOL. Ltd., Tokyo, Japan). LDL-cholesterol was computed according to Friedewald’s formula (Friedewald et al., 1972):

Statistical Analysis
The experimental data are presented as the means and standard errors of the means. Dunnett’s t-test for multi-comparison was applied to determine the statistical significance of the difference in effects among the three groups in the animal study, using the JMP software package, version 4.05J (SAS Institute, Japan), with the significance level set at P < 0.05. The paired Student’s t-test was conducted with Microsoft Excel, version 97, to determine the statistical significance of the difference in effects between the two groups in the human study.

	RESULTS

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Gastric Juice and Bile Tolerance
High tolerance of strain BL1 to gastric juice was observed. Incubation in artificial gastric juice at pH 3.0 and 37°C for 1.5 and 2.0 h yielded tolerance rates of 32.4 and 11.3%, respectively. The strain showed also a high tolerance to bile (Table 4). Multiplication of the strain in skim milk with or without bile was very slow, probably due to the restriction in nutrients. However, complete survival was observed after 5 h incubation in the presence of oxgall at 0.4 and 1.6%. More than half of the bacterial cells survived after 16 h incubation in 1.6% oxgall.

Human Study
At wk 4, all subjects completed the study. The mean BW were 69.0 ± 6.7 and 67.3 ± 7.5 kg for the bifidobacterium yogurt (B-) group and placebo yogurt (P-) group, respectively. Only minor fluctuation in BW, not related to treatment, was observed. Physical examinations of all subjects performed at the time of blood sampling showed no abnormalities.

Table 7 shows the average values of serum lipids before and after the experimental diet intake for the 32 subjects. No significant change was found in serum lipid concentration during the experimental period, for both the B-group and P-group, although there was a trend toward reduction in the B-group. When the 16 subjects in the B-group were analyzed individually, most showed reduced total cholesterol levels. Considering up to 5% as an acceptable variation according to the laboratory method, we found that in the B-group, five subjects did not vary (had a total cholesterol variation less than 5%), eight subjects showed a reduction (more than 5%), and three subjects showed an increase (more than 5%) of their cholesterol levels. On the other hand, there were 14 subjects without change, one subject with a reduction, and one subject with an increase in cholesterol level in the P-group.

	DISCUSSION

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The results of the present study demonstrate that, as compared to traditional yogurt fermented with ordinary lactic acid bacteria, the bifidobacterium-fermented yogurt was effective in improving serum lipids in rats as well as in humans with moderate hypercholesterolemia. In the animal experiments, in comparison with the control, bifidobacterium milk diets resulted in significantly lower levels of serum total cholesterol, LDL-cholesterol, and triglyceride, but SL milk brought about only slight, nonsignificant changes. A single-blind, parallel group study with bifidobacterium yogurt and placebo yogurt in healthy men (n = 16) indicated a reduction trend in serum total cholesterol after intake of the bifidobacterium yogurt. A significant reduction of serum cholesterol was observed among subjects with moderate hypercholesterolemia (serum total cholesterol > 240 mg/dl, n = 9).

The finding that bifidobacterium-fermented milk lowers serum total cholesterol concentrations in rats agrees with data from other studies involving various milk products containing selected strains of lactic acid bacteria. Akalin et al. (1997) demonstrated that yogurt fermented with L. acidophilus significantly reduced the serum total cholesterol concentration in mice, whereas yogurt fermented with ordinary yogurt starters containing S. thermophilus and L. delbrueckii subsp. bulgaricus did not. In experiments in rats, Beena and Prasad (1997) demonstrated that bifidus yogurt fortified with condensed whey lowered the cholesterol level more than did ordinary yogurt fortified with condensed whey. Reductions of serum total cholesterol and triglycerides were observed with selected strains including L. acidophilus (Danielson et al., 1989), L. reuteri (Taranto et al., 1998, De Roos et al., 1999), L. casei (Hashimoto et al., 1999), and L. gasseri (Usman and Hosono, 2000) in experimental animal studies.

The present animal studies indicated a slight, nonsignificant decrease in the concentration of HDL-cholesterol but a significant decrease in the concentrations of LDL-cholesterol in the group fed bifidobacteria in comparison with the control group. Because the LDL-cholesterol was calculated from values for serum total cholesterol, triglycerides, and HDL-cholesterol using the equations of Friedewald et al. (1972), the reduction in serum LDL-cholesterol concentrations would be expected. The present results related to HDL- and LDL- cholesterol generally agree with other findings (Aklain et al., 1997; Danielson et al., 1989; De Smet et al., 1998), although a decrease in total cholesterol was found with a simultaneous increase of HDL-cholesterol in some studies (Hashimoto et al., 1999; Taranto et al., 1998).

The mechanisms of serum lipid improvement have been suggested from in vitro and in vivo studies. In vitro experiments demonstrated that intestinal lactic acid bacteria have the capacity to assimilate and bind cholesterol as well as bile acids with the bacterial cells (Bottazzi et al., 1986; Gilliland et al., 1985, Hosono and Tono-oka, 1995). From these results, it is conceivable that serum total cholesterol is reduced by inhibiting absorption in the intestine as a result of the assimilating and binding of cholesterol as well as bile acids by the lactic acid bacteria. Promotion of bile acid excretion due to deconjugation to produce free bile acids was proposed as another mechanism for serum lipid improvement (Grundy, 1972). It is known that the serum cholesterol level decreases when a component suppresses resorption of bile acid in the intestine because the catabolism of cholesterol is promoted in the liver. Binding of cholesterol or bile acids was not investigated in the present studies, but a higher excretion of bile acids in feces, although not statistically significant (P = 0.068), was observed in the group fed bifidobacterium milk. Increased fecal output of neutral sterols and bile salts was observed in piglets fed L. reuteri (De Smet et al., 1998).

Surviving passage through the gastrointestinal tract is believed to be important for probiotics to function in the intestine (Gilliland and Walker, 1990; Salminen and von Wright, 1993). Very few studies have mentioned the survival of the bacterial cells supplemented to the diets in animal experiments. We noticed that lyophilized cells of lactobacilli and bifidobacteria died largely within 1 to 2 d after being combined with the diet, presumably due to the moisture of the diet. In the present studies, special attention was given to maintaining a higher survival of the bacterial cells by means such as employing dried diet materials, mixing the cells into diets just before administration, and replacing the diets daily.

B. longum strain BL1 was shown to survive at a high rate in artificial gastric juice at pH 3.0 for 1.5 to 2 h. Bacteria would contact pH values ranging from 2.0 to 8.0 in the stomach, depending upon whether or not food was being consumed (Hood and Zottola, 1988). Resisting exposure to pH 3 for 1.5 to 2 h is considered one standard for low-pH tolerance of probiotic bacteria (Itoh, 1992). Surviving bacteria from the stomach would then contact bile in the small intestine. Multiplication of bifidobacteria in the small intestine should not be expected since the bacteria are obligately anaerobic (Mitsuoka, 1978). Tolerance to bile is thus a criterion for bifidobacteria to survive passage through the small intestine. The present strain was found to survive at a relatively high concentration of bile for 5 to 16 h, which is assumed to be the usual time for food to pass through the small intestine. However, S. thermophilus and L. delbrueckii subsp. bulgaricus, the two most important organisms used for yogurt manufacture, were not actually observed in the intestinal tract because of their low tolerance for bile salts, low resistance to acid pH, and rather selective requirements for sugars (Harvenaar and Minekus, 1996; Rasic and Kurmann, 1979). Strain BL1 was found to possess a strong bile salt hydrolase activity (unpublished results). The surviving cells of strain BL1 may thus function in cholesterol reduction probably by binding with cholesterol and bile acids, as well as by suppressing bile acid resorption by deconjugation.

It was noticed that intake of fermented milk does not always show similar effects in humans as in animals because of differences between species in the cholesterol metabolism mechanism (Friedewald et al., 1972; Kawase et al., 2000). Studies in humans, however, have resulted in contradictory outcomes. Hepner et al. (1979) carried out the first randomized crossover trial with 17 subjects and indicated a 5 to 9% lowering effect of serum total cholesterol by unpasteurized yogurt. Randomized, double-blind, and placebo controlled studies also demonstrated significant reductions in serum total cholesterol and LDL-cholesterol by a milk product fermented with Enterococcus faecium and two strains of S. thermophilus (Agerbkæk et al., 1995; Richelsen et al., 1996). Kiessling et al. (2002) observed that consumption of yogurt increased the serum concentration of HDL-cholesterol and lead to the desired improvement of the LDL/HDL cholesterol ratio. Kawase et al. (2001) demonstrated a suppressing effect on cholesterol uptake by a fermented milk supplemented with S. thermophilus in humans. However, studies employed fermented milks supplemented with L. acidophilus as well as other ordinary lactic acid bacteria resulted in lack of any effect (De Roos et al., 1999; Thompson et al., 1982). Studies conducted in human subjects are thus ambiguous regarding the effects of fermented dairy products on serum cholesterol concentrations. Further, although there were observations concerning in vitro cholesterol assimilation as well as cholesterol-lowering effect in animal experiments for bifidobacteria (Tahri et al., 1995; Beena and Prasad, 1997), hypocholesterolemic effect of bifidobacteria has not been demonstrated on human. To verify the effect of the bifidobacterium yogurt on human serum lipids as observed on rats, we carried out the present pilot human study. The bifidobacterium yogurt and the placebo yogurt have the same contents of milk fats, proteins, carbohydrates, and energy. The only difference was the presence or absence of the targeted bifidobacteria. A reduction trend in serum total cholesterol concentration was observed in the group of volunteers who consumed bifidobacterium yogurt, but the change was not significant. A significant reduction of total cholesterol after the 4 wk intake of the bifidobacterium yogurt was found for those subjects with initial total cholesterol values over 240 mg/dl (n = 9, P < 0.05). We have no obvious explanation for the more notable effect in subjects with moderate hypercholesterolemia (serum total cholesterol > 240 mg/dl) than those with serum total cholesterol in the range of 220 to 240 mg/dl. This could perhaps be attributable to the small numbers of subjects in each group and the relatively high variation that occurred in the values of some subjects within each group. We could not exclude the influence of personal daily lifestyle on the changes of serum lipid levels, because each subject was advised to maintain ordinary living habits without great restriction during the experimental period. However, it should be noted that variability in effectiveness is commonly seen with lipid-lowering agents (Lees et al., 1977). Asaoka et al. (1996) also reported a selective lowering effect on serum total cholesterol in adult men administered a drink containing depolymerized sodium alginate, in which lowering was observed in those subjects with an initial total cholesterol level over 220 mg/dl, but not in those with a normal level (<220 mg/dl).

Correlated reduction of LDL- and HDL-cholesterol was observed along with the decrease in total cholesterol, but statistical significance occurred neither for the whole group nor for the portion with moderate hypercholesterolemia (serum total cholesterol <240 mg/dl). The related changes of total cholesterol, LDL-cholesterol, and HDL-cholesterol may support the hypothesis that the strain acts by promoting excretion of cholesterol and bile acid, rather than by affecting hepatic cholesterol synthesis.

The present results indicate the potential of the bifidobacterium yogurt in lowering serum total cholesterol, especially in those subjects with moderate hypercholesterolemia (serum total cholesterol >240 mg/dl). The borderline for the onset of medical therapy for serum total cholesterol has been recently raised from 220 to 240 mg/dl in Japan. The clinical importance of this yogurt is thus noteworthy, and research is being planned with more subjects and for longer terms in combination with nutrition instruction for further clarification.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The present study demonstrated that, as compared to traditional yogurt fermented with ordinary lactic acid bacteria, bifidobacterium yogurt fermented with a probiotic strain of Bifidobacterium longum BL1 and a drinking yogurt fermented with strain BL1 plus ordinary lactic acid bacteria were effective in improving serum lipids in rats and in humans with initial serum cholesterol level more than 240 mg/dl, respectively. B. longum strain BL1, which possesses a high tolerance to gastric juice and bile acids and shows a strong bile salt hydrolase activity, is assumed to survive passage through the gastrointestinal tract and function in cholesterol reduction. These results indicate the potential of the probiotic effect of the Bifidobacterium fermented products on serum cholesterol reduction.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
We wish to thank Dr. M. Takase and Dr. T. Yaeshima of the Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd., for their helpful advice and technical assistance.

Corresponding author:
J.Z. Xiao; e-mail:
j_xiao{at}morinagamilk.co.jp.

Received for publication November 1, 2002. Accepted for publication February 26, 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 


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