Effects of fermented milk peptides supplement on blood pressure and vascular function in spontaneously hypertensive rats

This study investigated the effects of fermented milk peptides (FMP) in spontaneously hypertensive rats (SHR). Systolic blood pressure (SBP) and the angiotensin-converting enzyme (ACE) activity were significantly ameliorated in FMP (10 mg/kg BW/day) group compared to those of the skimmed milk or captopril (CAP, 50 mg/kg BW/day) groups, respectively. The higher plasma nitric oxide (NO) level and the reduced aortic thickness were found in the FMP and CAP groups versus those in the control group, respectively. In conclusion, fermented milk products containing tripeptides have been shown to be a well-tolerated dietary option against mild hypertension.     

富含ACE抑制肽的酪蛋白水解物及其功能性酸奶对SHR血压的影响

目的:用酪蛋白双酶(胃蛋白酶和胰蛋白酶)水解物(A)及其超滤物(截留分子量6ku)(B)、水解物的脱苦产物(C)及其超滤物(D)分别灌胃12周龄雄性原发性高血压大鼠(SHR)和正常Wistar大鼠,研究其降血压作用。方法:A和C的灌胃剂量分别为0.083、0.25、0.75g/kgmb;B和D的灌胃剂量分别为0.01、0.03、0.09g/kgmb;检测灌胃后8h内大鼠尾动脉收缩压(SBP)的变化。结果:四种物质的高剂量对正常Wistar大鼠的血压无影响;四种物质的三种剂量分别灌胃SHR大鼠对其血压有明显的降低作用,其中A(0.25g/kgmb)、C(0.75g/kgmb)、B(0.03g/kgmb)、D(0.03g/kgmb)灌胃SHR大鼠在第4h达到最高降压幅度,分别为22.630、19.375、33.375、29.000mmHg。用两种超滤物制备发酵乳灌胃SHR大鼠在第4h血压分别下降了36.875、31.875mmHg;且间隔4h连续灌胃两种发酵乳可以在8h内维持较低的血压。结论:利用富含ACE抑制肽的酪蛋白水解物制备的功能性发酵乳在控制和治疗高血压方面有一定的效果,这为以后食源性降压食品的开发提供了一定的依据。      

两种血管紧张素转化酶抑制肽作用于靶标的分子机理

采用脱脂乳为原料,利用四步反相高压液相色谱从Lactobacillus helveticus 和Lactobacillus casei subsp. casei制作的酸乳中分离到两种血管紧张素转化酶Ⅰ(angiotensin Ⅰ -converting enzyme,ACE)抑制肽VPP 和IPP,测得它们抑制ACE 活性的IC50 分别为8.89μmol/L 和5.17μmol/L。根据氨基酸序列分析的结果,构建VPP 和IPP 的分子结构,通过分子柔性对接方法研究它们与ACE 相互作用的分子机理,确定它们的作用位点、作用力类型及相互作用能。结果表明：VPP、IPP 和ACE 的活性口袋之间均形成3 个氢键,且存在疏水,亲水等作用力,IPP 与ACE之间的结合较VPP 更稳定,与IPP 比VPP 具有较低的IC50 值的实验结果相一致。     

Casein-derived bioactive tripeptides Ile-Pro-Pro and Val-Pro-Pro attenuate the development of hypertension and improve endothelial function in salt-loaded Goto–Kakizaki rats

Increased blood pressure and plasma cholesterol concentration are the major risk factors for cardiovascular diseases. We have previously shown that fermented milk products containing casein-derived bioactive tripeptides, isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) partly inhibit the blood pressure increase in spontaneously hypertensive rats (SHR). In the present study, we investigated the effect of bioactive tripeptide-containing fermented milk products (with or without plant sterols) on blood pressure and vascular function in salt-loaded type 2 diabetic Goto–Kakizaki (GK) rats after 8 weeks’ treatment. The development of blood pressure was attenuated in the groups receiving tripeptide-containing products (?10 to ?12 mmHg vs. the control product group, P < 0.01). Tripeptide-containing products enhanced the impaired endothelium-dependent relaxation of mesenteric arteries. Also the role of endothelium-derived hyperpolarizing factor (EDHF) was demonstrated. Interestingly, pretreatment of arterial rings with verapamil normalized the impaired endothelial function. Endothelium-independent relaxations were similar between the groups. Furthermore, tripeptides had positive effects on renin–angiotensin–aldosterone system.     

产ACE抑制肽乳酸菌的筛选、益生特性评定及应用

目的:筛选出富产血管紧张素转化酶(Angiotensin converting enzyme,ACE)抑制肽的乳酸菌并评定其益生特性.方法:依据蛋白水解度及ACE抑制率对乳酸菌进行初筛,然后依据模拟胃肠消化后的ACE抑制率最终筛出2株乳酸菌ZJUIDS09和ZJUIDS11,进一步评价其耐酸、耐胆盐、抗生素耐药性和抑菌...     

Repressive processing of antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, in Lactobacillus helveticus fermented milk by added peptides

Lactobacillus helveticus can release the antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), from casein in fermented milk by a specific proteolytic system. To better understand the regulation of gene expression of the proteolytic enzymes thought to link to the processing of both antihypertensive peptides in L. helveticus, microarray analysis for whole gene expression in the presence and absence of added peptides in the fermented milk was studied. The productivity of both VPP and IPP in L. helveticus CM4 fermented milk was repressed by adding 2% quantity of Peptone as peptide mixture to the milk. Among the selected 13 amino acids, Gly, Ile, Leu, Phe, Met, Ser and Val were effective in the repression of the productivity of VPP and IPP in the fermented milk. The activity of the cell wall-associated proteinase, which may play a key role in the processing of the two antihypertensive peptides, was significantly repressed by the addition of the 2% quantity of Peptone into the fermented milk. By DNA microarray analysis it was found that prtH2 corresponding to the cell wall-associated proteinase gene, most of the endopeptidase genes such as pepE, pepO1, pepO2 and pepO3, most of the oligopeptide transporter genes, such as dppA2, dppB, dppC, dppD and dppF, most likely involved in the processing of VPP and IPP were down-regulated. These results suggest that amino acids released from milk peptides in the fermented milk might down-regulate the gene expressions of some of the proteolytic enzymes and may cause repression of the release of VPP and IPP in L. helveticus fermented milk.     

Novel probiotic-fermented milk with angiotensin I-converting enzyme inhibitory peptides produced by Bifidobacterium bifidum MF 20/5

In previous research, we have demonstrated that Bifidobacterium bifidum MF 20/5 fermented milk possessed stronger angiotensin converting enzyme (ACE) inhibitory activity than other lactic acid bacteria, including Lactobacillus helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP). The aim of this study is to investigate the ACE-inhibitory peptides released in B. bifidum MF 20/5 fermented milk. The novel ACE-inhibitory peptide LVYPFP (IC₅₀ = 132 μM) is reported here for the first time. Additionally, other bioactive peptides such as the ACE-inhibitor LPLP (IC₅₀ = 703 μM), and the antioxidant VLPVPQK were identified. Moreover, the peptide and amino acid profiles, the ACE-inhibitory activity (ACEi), pH, and degree of hydrolysis of the fermented milk were determined and compared with those obtained in milk fermented by L. helveticus DSM 13137. The sequences of the major bioactive peptides present in fermented milk of B. bifidum and L. helveticus were identified and quantified. B. bifidum released a larger amount of peptides than L. helveticus but no IPP or VPP were detected in B. bifidum fermented milk. Also the lactotripeptide concentrations and ACEi were higher in L. helveticus fermented milk when the pH was maintained at 4.6. This may represent a technical advantage for B. bifidum that reduces the pH at a slow enough rate to facilitate the peptide generation without the need for pH control. Thus these findings show the potential for the use of this probiotic strain to produce fermented milk with a wider range of health benefits including reduction of blood pressure.     

Hot topic: Changes in angiotensin-converting enzyme inhibition and concentrations of the tripeptides Val-Pro-Pro and Ile-Pro-Pro during ripening of different Swiss cheese varieties

The angiotensin-converting enzyme (ACE) inhibitory activity and the concentration of the 2 ACE-inhibiting tripeptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) were studied during cheese ripening in 7 Swiss cheese varieties. The semi-hard cheeses Tilsiter, Appenzeller 1/4 fat, Tête de Moine, and Vacherin fribourgeois and the extra-hard and hard cheeses Berner Hobelkäse, Le Gruyère, and Emmentaler were investigated. Three loaves of each variety manufactured in different cheese factories were purchased at the beginning of commercial ripeness and investigated at constant intervals until the end of the usual sale period. Good agreement was found between ACE-inhibitory activity and the total concentration of VPP and IPP at advanced ripening stages. In most of the investigated varieties ACE-inhibitory activity and the concentration of the 2 tripeptides initially increased during the study period. A decline in the concentration of VPP and IPP was obtained toward the end of the investigated period for Tilsiter and Gruyère. The ratio of VPP/IPP decreased during ripening in all varieties with the exception of Emmentaler. However, large variations were observed among the cheese varieties as well as the individual loaves of the same variety. Chemical characterization of the investigated cheeses revealed that qualitative differences in the proteolysis pattern, not quantitative differences in the degree of proteolysis, are responsible for the observed variations in the concentrations of VPP and IPP. The presence of Lactobacillus helveticus in the starter culture was associated with elevated concentrations of VPP and IPP. The results of the present study show that concentrations of VPP and IPP above 100 mg/kg are attainable in semi-hard cheese varieties after ripening periods of about 4 to 7 mo and that stable concentrations of the 2 antihypertensive tripeptides can be expected over several weeks of cheese ripening.     

Antihypertensive activity of milk fermented by Enterococcus faecalis strains isolated from raw milk

A total of 231 microorganisms were isolated from raw cow milk samples and the angiotensin-converting enzyme-inhibitory (ACEI) activity of the resultant fermented milk produced with the isolated microorganisms was assayed. Forty-six of these microorganisms were selected on the basis of high ACEI activity. Four Enterococcus faecalis strains stood out as producers of fermented milk with potent ACEI activity (IC₅₀ (the protein concentration that inhibits 50% of ACE activity): 34–59 μg mL⁻¹). Single doses (5 mL kg⁻¹) of the whey fraction obtained from these fermented milk samples were administered to spontaneously hypertensive rats (SHR) and to normotensive Wistar-Kyoto (WKY) rats in order to investigate their possible antihypertensive activity. Highly significant decreases in the systolic blood pressure (SBP) and in the diastolic blood pressure (DBP) were observed when the fermented milk was administered to SHR. Nevertheless, the fermented milk did not modify the SBP and the DBP of the WKY rats. Raw cow milk is an excellent source of wild lactic acid bacteria able to produce fermented milk with antihypertensive activity and antihypertensive activity of milk fermented by Enterococcus faecalis strains was associated with peptides different from Ile-Pro-Pro and Val-Pro-Pro.     

Antihypertensive activity of casein-enriched milk fermented by Lactobacillus helveticus

Milk proteins contain peptidic angiotensin I-converting enzyme (ACE) inhibitors, which can be released by proteolysis during milk fermentation by some strains of Lactobacillus helveticus. Reconstituted milk media containing skim milk powder (12%), skim milk powder (10%) with added sodium caseinate (5%) or whey protein isolate (5%) were fermented by L. helveticus strains R211 and R389, and further tested for bacterial growth, proteolysis (free NH₃ groups) and ACE-inhibitory activity. The antihypertensive activity in spontaneously hypertensive rats (SHR) was also investigated with caseinate-enriched milk unfermented (UFM) and fermented by the two strains of L. helveticus. Caseinate-enriched milk fermented by both strains showed higher proteolysis and ACE-inhibitory activity, indicating that ACE-inhibitory peptides are probably released from caseins during milk fermentation. Significant decreases in mean arterial blood pressure in SHR rats were measured following oral administration of UFM milk at doses of 1.0 and 2.5 g kg⁻¹ of body weight, and milk fermented by R211 or R389 strains at doses of 0.5, 1.0 and 2.5 g kg⁻¹ of body weight. The antihypertensive activity of UFM could be explained by the release of ACE-inhibitory peptides from caseins during the digestion process.     

瑞士乳杆菌H11发酵乳饮料的ACE抑制活性及代谢组学研究

目的:研究瑞士乳杆菌H11与副干酪乳杆菌Lc-01两种发酵乳饮料在贮藏期间的代谢差异变化。方法:使用气相色谱-质谱（SPME-GC-MS）联用、高效液相色谱（HPLC）和超高效液相色谱串联四级杆飞行时间质谱（UPLC/Q-TOF MS）技术对4 ℃、贮藏28 d期间发酵乳饮料中的挥发性风味物质、代谢物以及ACE抑制活性之间的差异进行分析。结果:在4 ℃贮藏28 d后,瑞士乳杆菌H11发酵乳饮料的体外ACE抑制活性比副干酪乳杆菌Lc-01高60%以上,ACE抑制肽VPP和IPP含量也显著高于Lc-01（P<0.05）。采用SPME-GC-MS发现瑞士乳杆菌H11发酵乳饮料中香气成分丰富,特征风味物质2-庚酮和2-壬酮相对含量较高,分别为43.84%和12.39%。基于UPLC/Q-TOF MS的结果表明,贮藏期间两种发酵乳饮料的主要代谢差异物为肽、氨基酸和有机酸。结论:瑞士乳杆菌H11在制备发酵乳饮料方面存在巨大潜力。     

Identification and characterization of novel angiotensin-converting enzyme inhibitors obtained from goat milk

The aim of the present study was to identify and characterize new inhibitory peptides of angiotensin I-converting enzyme (ACE) from goat milk and to analyze the effect of long-term intake of a goat milk hydrolysate-supplemented (GP-hyd) diet on the development of hypertension in spontaneously hypertensive rats (SHR). Three new inhibitory peptides for ACE (TGPIPN, SLPQ, and SQPK) were isolated. The inhibitory concentration 50% (IC₅₀) values of individual peptides were 316, 330, and 354 μmol/L, respectively. Only TGPIPN was found to pass intact a monolayer of Caco-2 cells in small amounts. The SHR fed for 12 wk a diet (GP-hyd) enriched in a hydrolysate containing these peptides (estimated intake of TGPIPN was 230 mg/kg per d) showed lower (approximately 15 mmHg) systolic blood pressure than animals fed a control diet. The ACE activities in the aorta, left ventricle, and kidney were significantly decreased in the GP-hyd group compared with those of the control group and were similar to those found in SHR fed captopril (130 mg/kg per d). Impaired endothelium-dependent relaxation to acetylcholine by aortic rings from SHR was improved in those fed the GP-hyd diet. The left ventricle weight and kidney weight index were significantly reduced in the GP-hyd group and captopril groups. Moreover, long-term treatment of SHR with a diet enriched in goat milk hydrolysate, or captopril, attenuated the development of hypertension, cardiac and renal hypertrophy, and endothelial dysfunction. These effects might be related to the in vivo inhibitory effects of the hydrolysate on tissue ACE activity.     

紫薯水溶性提取物以及紫薯酸奶对SHR血压的影响

以紫薯为原料,12周龄雄性原发性高血压大鼠(SHR)和正常Wistar大鼠为受试动物,研究紫薯水溶性提取物的降血压作用。用紫薯水溶性提取物以低、中、高3个剂量(0.12、0.6、1.2g/kg·bw)灌胃SHR大鼠和正常Wistar大鼠,观察对大鼠尾动脉收缩压(SBP)的影响。结果表明:单次灌胃8h内,低、中、高三个剂量对正常Wistar大鼠的血压无影响;三种剂量对SHR的血压有明显的降低作用,且中、高剂量作用效果较好。灌胃后,中剂量在2h时达到最低值,下降了27.25mmHg,高剂量在4h时下降了约33.00mmHg。连续灌胃三种剂量水溶性提取物可较长时间维持低水平SBP。用上述三种不同剂量制备紫薯酸奶,灌胃SHR大鼠。单次灌胃三种酸奶都有降血压作用,低剂量组在4h时达到最低值,下降了20.25mmHg,中剂量组在3h时达到最低值,下降了25.13mmHg,高剂量组在4h时达到最低值,下降了28.13mmHg,且连续灌胃紫薯酸奶可以在8h内维持较低的血压。      

Development of a salmon protein hydrolysate that lowers blood pressure

A salmon protein hydrolysate (SPH) was developed containing several angiotensin I-converting enzyme (ACE) inhibitory tripeptides the most abundant of which were Val-Leu-Trp, Val-Phe-Tyr, and Leu-Ala-Phe. Simulated digestion experiments showed that active constituents of SPH would survive in the digestive tract and be available for absorption into the bloodstream. In fact, ACE inhibitory activity was improved following simulated digestion suggesting that there were larger peptides in SPH that might contribute to bioactivity in vivo. A single oral dose (1,500 mg/kg body mass) of SPH significantly lowered blood pressure in spontaneously hypertensive rats (SHR). The treatment of SHR with either SPH fraction (<3,000 Da) or SPH fraction (>3,000 Da) reduced blood pressure. We conclude that the ability of SPH to lower blood pressure is due to a combination of ACE inhibitory tripeptides as identified, as well as additional unknown, peptide species that are generated during digestion of SPH in the gastrointestinal tract.     

Antihypertensive effect of bioactive peptides produced by protease-facilitated lactic acid fermentation of milk

Milk was fermented for up to 5 h at 43 °C with two lactic acid bacteria (Streptococcus thermophilus, Lactobacillus bulgaricus). A protease, flavourzyme, was added at the beginning of fermentation. The whey fraction was separated from the fermented milk and freeze-dried. During the 5 h of fermentation, the soluble protein content increased from 4.9 to 57.4 mg/g and peptide content increased from 2.1 to 32.8 mg/g, while inhibition of angiotensin I-converting enzyme (ACE) increased by a decrease of IC₅₀ from 0.708 to 0.266 mg/ml, respectively. The whey was fractionated into four fractions by size exclusion chromatography on a Sephadex G-15 column. The fourth fraction of the whey showed the highest inhibitory efficiency ratio (IER) being 1329%/mg/ml. The amino acid sequence of the inhibitory peptide was Tyr-Pro-Tyr-Tyr, of which the IC₅₀ was 90.9 μM. The whey showed mixed-type inhibition kinetics, while Captopril, the positive control showed competitive inhibition on ACE. Their K_i values were 0.188 mg/ml and 0.0067 μg/ml, respectively. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) was reduced to 15.9 and 15.6 mm Hg, respectively, in spontaneously hypertensive rat (SHR), after 8 weeks of oral administration of diluted whey (peptide concentration 4.9 mg/ml).     

Angiotensin-converting enzyme activity in plasma and tissues of spontaneously hypertensive rats after the short- and long-term intake of hydrolysed egg white

This paper evaluates the effects of the short- (1 g/kg) and long-term (0.5 and 1 g/kg/day) oral intake of egg white hydrolysed with pepsin (hEW) and the long-term oral intake (1 g/kg/day) of egg white (EW) on local angiotensin-converting enzyme (ACE) activities in plasma and other tissues of spontaneously hypertensive rats (SHR), as compared to the effect of the ACE inhibitor prototype captopril. The rats treated with hEW were classed in a different group than the control rats and the rats treated with EW by cluster analysis, taking into account their tissue ACE activities and their systolic blood pressure (SBP). Principal component analysis (PCA) showed that SBP in SHR was negatively related with ACE activity in plasma and positively related with ACE activity in aorta and kidney. ACE activity in plasma significantly increased after the long-term treatment with hEW (0.5 g/kg/day). ACE activity in aorta and kidney was significantly inhibited 4 h after the short-term administration of hEW. The long-term treatment with hEW caused local effects on ACE activity in aorta, kidney and lungs that followed a pattern similar, but less pronounced, than that caused by captopril.     

Vascular effects and antihypertensive properties of κ-casein macropeptide

The blood-pressure-lowering effect of bovine κ-casein macropeptide (CMP), previously reported to exhibit a modest angiotensin-converting enzyme (ACE)-inhibitory activity in vitro, was evaluated in spontaneously hypertensive rats (SHR). The oral administration of CMP (150 mg kg⁻¹) significantly reduced the systolic blood pressure (SBP) of SHR. The antihypertensive action was more pronounced when CMP was treated with trypsin. The sequence MAIPPKK, identified in the tryptic hydrolysate of bovine CMP, significantly reduced blood pressure at a dose of 10 mg kg⁻¹. The CMP and its tryptic peptides induced relaxation of endothelium-intact aortic rings. The sequence MAIPPKK also evoked a significant relaxation effect; however, the shorter sequence MAIPPK and the strong ACE-inhibitory tripeptide IPP exhibited a vascular relaxing effect lower than 10%. The implications of vascular relaxing mechanisms, as well as the possibility that the tripeptide IPP is at least partially responsible for the antihypertensive effects of CMP, are discussed.     

基于综合性Meta分析的乳三肽VPP和IPP在随机安慰剂对照实验中的抗高血压效能

为评估口服乳三肽VPP（Val-Pro-Pro）和IPP（Ile-Pro-Pro）对血压的影响,基于综合性Meta分析选择了包含在21 个研究中的27 项临床实验、2 142 个高血压前期或高血压患者。由于存在异质性,采用随机影响模式进行分析。结果表明：Meta分析中未发现发表偏倚,收缩压（systolic blood pressure,SBP）降低了1.78 mmHg（95%可信区间为－2.47～－1.08,P＜0.001）,舒张压（diastolic blood pressure,DBP）降低了0.67 mmHg（95%可信区间为－1.11～－0.23,P＜0.001）。亚组分析结果表明：亚洲人群SBP为－6.56 mmHg、DBP为－3.01 mmHg,2008年前的研究对象SBP为－6.57 mmHg、DBP为－3.08 mmHg,口服乳三肽剂量＜5 mg/d的患者SBP为－5.20 mmHg、DBP为－2.05 mmHg,以及诊所偶测SBP（－2.74 mmHg）与DBP（－1.18 mmHg）和家庭自测SBP（－7.61 mmHg）与DBP（－4.83 mmHg）都显著下降,而相应的比较对象则没有。因此,包含乳三肽VPP和IPP的功能性食品对高血压前期或轻度高血压患者是有效的,特别是在低剂量食用时对亚洲人群的诊所偶测血压和家庭自测血压有明显效果。     

Structural analysis of a new anti-hypertensive peptide (beta-lactosin B) isolated from a commercial whey product

Angiotensin-converting enzyme (ACE) inhibitory activities and anti-hypertensive activities in spontaneously hypertensive rats (SHR) of 12 kinds of commercial peptides of food additive grade were measured. Four peptide products derived from milk proteins showed strong anti-hypertensive activities (>-18.0 mm Hg). A sample of WE80BG derived from whey proteins showed the strongest anti-hypertensive activity (-21.2 +/- 16.9 mm Hg) with a medium level of ACE inhibitory activity (53.6%), and it was subjected to hydrophobic and gel filtration chromatography. From the low molecular weight fraction, an anti-hypertensive peptide was isolated by using reversed-phase HPLC, and it was found to be a tetrapeptide, alanine-leucine-proline-methionine (Ala-Leu-Pro-Met, ALPM), the origin of which was estimated to be beta-lactoglobulin f 142 to 145. At 8 h after oral administration of ALPM in SHR, systolic blood pressure was significantly decreased (-21.4 +/- 7.8 mm Hg), but the IC50 value (concentration of peptide needed to inhibit 50% of the ACE activity) of ALPM was not so high. We named the Ala-Leu-Pro-Met "beta-lactosin B." This peptide is the second anti-hypertensive peptide found from beta-lactoglobulin. Because WE80BG containing ALPM was also found to show the strongest anti-hypertensive activity (-24.5 +/- 10 mm Hg) at 8 h after oral administration in SHR, WE80BG would be suitable for application to the development of a new food expected to have anti-hypertensive effects.     

Serum Cholesterol Levels in Rats Fed Skim Milk Fermented by Lactobacillus Acidophilus

The effects of reconstituted skim milk, and the same fermented by Lactobacillus acidophilus, were tested in rats. Rats were fed a stock diet and drinking water containing one of three milk treatments: (1) no supplementary milk; (2) 10% milk; or (3) 10% milk fermented by L. acidophilus. After 4 wk, rats receiving the fermented milk had lower (P < 0.05) serum cholesterol levels (65 mg/dl) than did the water-fed (78 mg/dl) or milk-fed (79 mg/dl) rats. Weight gain, feed intake, liver lipid contents and fecal lactobacilli counts were not different among treatment groups. Data indicate that factors influencing serum cholesterol levels were produced during fermentation of the milk.