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* Corresponding author. Dipartimento di Protezione delle Piante e Microbiologia
*通讯作者。

网络地址的电子microbiologiaprotezione迪
Applicata, University of Bari, via Amendola 165/a, 70126 Bari, Italy. Tel.: þ39 80 544
applicata，巴里大学附近，通过165/70126，巴里，意大利。

电话：þ3980544 2948; fax: þ39 80 544 2911.
2948；传真：þ39805442911。

E-mail address: f.minervini@agr.uniba.it (F. Minervini).
电子邮件地址：f.minervini@agr.uniba.it（F .梅诺菲尼）。

Contents lists available at ScienceDirect
内容列表可在科学
Food Microbiology
食品微生物学
journal homepage: /locate/fm
网页：/locate/fm杂志
0740-0020/$ – see front matter 2009 Elsevier Ltd. All rights reserved.
0740-0020/ $–看到前面的问题2009爱思唯尔公司保留所有权利。

doi:10.1016/j.fm.2009.03.008
内政部：10.1016/j.fm.2009.03.008
自动检测语言中→ 英英→ 中中→ 日日→ 中翻译结果（英> 中）复制结果双语对照查看
Fermented goats’ milk produced with selected multiple starters as a potentially
发酵山羊'牛奶选用多首先作为一个潜在的
functional food
功能性食品
Fabio Minervini a,*, Maria Teresa Bilancia b, Sonya Siragusa a, Marco Gobbetti a, Francesco Caponio b
法比奥梅诺菲尼，*，玛丽亚特蕾莎bilancia乙，索尼娅席拉哥沙，马可·高贝蒂，弗朗西斯科卡波尼奥乙
a Dipartimento di Protezione delle Piante e Microbiologia Applicata, University of Bari, Bari, Italy
一个网络地址的迪protezione电子microbiologiaapplicata，巴里大学，巴里，意大利
b Dipartimento di Progettazione e Gestione dei Sistemi Agro-zootecnici e Forestali, Sezione Industrie Agro-alimentari, University of Bari, Bari, Italy
乙部设计电子受过系统agro-zootecnici电子绿洲林业庭，工业agro-alimentari，巴里大学，巴里，意大利
a r t i c l e i n f o
一个是我我我的啊
Article history:
文章历史：
Received 4 September 2008
4九月2008
Received in revised form
在修订的形式
13 January 2009
13一月2009
Accepted 21 March 2009
21月2009
Available online 27 March 2009
27月2009在线
Keywords:
关键词：
Fermented goats’ milk
发酵山羊奶
GABA
氨基丁酸
ACE-inhibitory activity
ACE抑制活性
Functional
功能
自动检测语言
自动检测语言中→ 英英→ 中中→ 日日→ 中翻译结果（英> 中）复制结果双语对照查看
a b s t r a c t
一个共和
A screening among five lactic acid bacteria, used alone or in combination, led to select a mixed starter
筛选五乳酸菌，单独使用或组合使用，导致选择混合起动
(Streptococcus thermophilus CR12, Lactobacillus casei LC01, Lactobacillus helveticus PR4, Lactobacillus
（嗜热链球菌Cr 12，干酪乳杆菌lc01pr4，瑞士乳杆菌，乳酸菌
plantarum 1288) capable to prod uce a fermented goats’ milk containing
g-aminobutyric acid (GABA) and
1288）能够产生发酵羊奶含有g -氨基丁酸（γ-氨基丁酸）和
angiotensin-I converting enzyme (ACE)-inhibitory peptides. The fermented milk was characterized by
血管紧张素转换酶（酶）抑制肽。

发酵乳的特点是
cell counts of lactic acid bacteria not lower than 7.0 log cfu g 1, even after 45 days of storage at 4 C.
细胞计数的乳酸菌不低于7日志祖克1，甚至在45天的存储在4C Fermentation of goats’ milk resulted in the production of ca. 28 mg kg 1 of GABA. Furthermore the
发酵的山羊'牛奶导致生产约28毫克公斤1丁酸。

此外，
fermented goats’ milk had an in vitro ACE-inhibitory activity of ca. 73%. Prolonged cold storage did not
发酵山羊奶有体外ACE抑制活性约73%。

长期冷藏不
significantly affect both the concentration of GABA and the ACE-inhibitory activity. Moreover, the taurine
显着影响的浓度的丁酸和ACE抑制活性。

此外，牛磺酸
content did not significantly vary during both fermentation and the entire storage period.
内容没有显着不同，在发酵和整个贮藏期。

2009 Elsevier Ltd. All rights
2009爱思唯尔公司版权所有
1. Introduction
1。

景区简介
During the last decade, interest of industries and consumers for
在过去十年中，工业和消费者的利益
functional foods has been exponentially increasing. According to
功能性食品已成倍增加。

根据
the Consensus Document issued by the European Concerted Action 协商一致的文件发出的欧洲协调行动
on Science of Functional Foods, a food may be referred to as
科学的功能性食品，食品可称为
‘‘functional’’, if it has been unequivocally proven that it positively
―'functional' '，如果它已经明确地证明，它积极
influences one or more biological functions in the human body,
影响一个或多个生物功能在人体，
improving the state of health and wellness, and reducing the risk to 改善健康状况和健康，并减少风险
develop a disease (Diplock et al., 1999). The use of milk with
发展一种疾病（迪普洛克等人。

，1999）。

用牛奶
particular nutritional properties (e.g., goats’, mares’ and donkeys’
特殊营养特性（例如，山羊，马和驴的
milk), alone or in combination with bacterial strains having probiotic 牛奶），单独或结合细菌菌株具有益生菌
properties and/or producing physiologically active metabolites,
性能和/或生产生理活性代谢产物，
represents one of the technology options for manufacturing
是一个技术方案的制造
new dairy functional beverages (Gomes et al., 1998).
乳品新功能饮料（戈麦斯等人。

，1998）。

Goats’ milk products, especially cheeses a nd yogurts, are very
山羊奶制品，特别是奶酪和酸奶，很
popular in the Mediterranean peninsula, Middle East, Southern
受欢迎的地中海半岛，中东，南部
Russia and Indian subcontinent (Tamime and Robinson, 1999). In 俄罗斯和印度次大陆（tamime和鲁滨孙，1999）。

在
the European Union, the value of production of g oats’ milk in 2005欧洲联盟，价值生产的山羊'牛奶2005
has been estimated 1371 million of euros (/
据估计，1371000000欧元（/
site/613/DesktopDefault.aspx?PageID¼613#ancor).
网站/613 /desktopdefault.aspx？pageid¼613#信息）。

Goats’ milk has be en described as having higher digestibility,
山羊奶已被描述为具有较高的消化率，
due to reduced dimensions of casein micelles and fat globules and 由于减少了尺寸的干酪素胶束和脂肪球
higher proportion of short to medium fatty acids (Park et al., 2007), 更高比例的短期和中期的脂肪酸（公园等。

，2007），
and lower al lergenic properties than cows’ milk (Spuergin et al.,
低过敏性比奶牛牛奶（spuergin等人。

，
1997). Besides, certain therapeutic properties in human nutrition, 1997）。

此外，某些治疗性质的人类营养，
such as a better utilization of fat and mineral salts in individuals
如更好地利用脂肪和矿物盐的人
suffer ing from malabsorption syndrome, are attributed to goats’
患有吸收不良综合征，是由于山羊
milk (Alferez et al., 2001). Goats’ milk contains also free taurine, one 牛奶（alferez等人。

，2001）。

山羊奶含有牛磺酸，一
of the final metabolic products of sulphur-containing amino acids
的最终代谢产物的含硫氨基酸
(Park et al., 2007), which may have several biological functions:
（公园等。

，2007），其中可能有若干生物学功能：
modulator of growth (Naismith et al.,1987) and of neuronal activity
生长调节剂（奈史密斯等人。

，1987）和神经元活动
(Haas and Hosli,1973; Huxtable,1989; Hussy et al.,1997; Jiang et al., （乙酸和hoslitable，1989，1973；；贱妇等人。

，1997；姜等。

，2004); conjugation of bile salts (Heuman and Bajaj, 1994); regulation 2004）；共轭胆汁盐（heuman和巴贾杰，1994）；调节
of osteoblast metabolism (Koide et al.,1999); protection of cells
成骨细胞的代谢（小出等人。

，1999）；细胞的保护作用
against various types of injury and prevention of cardiovascular
对不同类型的损伤和预防心血管疾病
damage (Warskulat et al., 2007); treatment of fatty liver of children
损伤（warskulat等人。

，2007）；治疗儿童脂肪肝
(Obinata et al., 1996).
（obinata等人。

，1996）。

The functional value of goats’ milk may be furt her exploited
功能价值的山羊'牛奶可进一步开发
through fermentation by selected microorganisms possessing
通过选择具有发酵微生物
specific features. A mixed starter comprising Lactobacillus acidophilus, 具体特点。

混合起动器包括嗜酸乳杆菌，
Bifidobacterium lactis and Streptococcus thermophilus has
乳双歧杆菌和嗜热链球菌有
been successfully used for fermentation of goats’ milk (Cutic
已成功用于发酵的山羊'牛奶（cutic
et al., 2004), and a high viability of probiotic strains in a fermented
等人。

，2004），和高活力的益生菌菌株在发酵
goats’ milk stored at 4 C for 10 days has been reported
山羊'牛奶储存在4摄氏10天已报告
(Kongo et al., 2006). Furthermore, the synthesis of folate from
（刚果等人。

，2006）。

此外，合成叶酸从
some lactic acid bacteria has been shown to occur during
有些乳酸菌已被证明发生在
fermentation of goats’ milk (Sanna et al., 2005), and the
发酵的山羊'牛奶（桑等人。

，2005），和
anti-atherogen ic effects of a goats’ milk fermented with the
抗动脉粥样硬化影响的山羊'牛奶发酵的
probiotic Lactobacillus fermentum ME-3 have been reported in 16
益生菌乳酸菌发酵me-3已报告16
healthy subjects (Kullisaar et al., 2003).
健康受试者（kullisaar等人。

，2003）。

Milk proteins are currently the main source of a range of
牛奶蛋白是目前的主要来源的范围
biologically active peptides. Many bioactivities are encrypted
生物活性肽。

许多生物活性是加密的
within the primary structure of milk proteins, requiring proteolysis 在主要结构蛋白的牛奶，需要蛋白质
for their generation from precursors. Proteolysis may
其产生的前体。

蛋白质可能
produce these biogenic peptides during food processing as well
产生这些生物肽在食品加工等
as during gastro-intestinal transit. Enzymes from different sources, 在肠道运输。

酶从不同来源，
including microbial enzymes, generate bioactive peptides
包括微生物酶，产生生物活性肽
during milk fermentation and cheese ripening, thereby enriching
发酵奶和奶酪成熟，从而丰富
the dairy products (Gobbetti et al., 2002). The capacity of several 乳制品（gobbetti等人。

，2002）。

能力的几个
lactic acid bacteria to synthesize angiotensin-I converting
乳酸细菌合成的血管紧张素转换
enzyme (ACE)-inhibitory peptides in dairy products is well
酶（酶）抑制肽奶制品是
known (Gobbetti et al., 2004; Muguerza et al., 2006; Donkor
已知（gobbetti等人。

，2004；muguerza等人。

，2006；唐科
et al., 2007). The type of lactic acid bacterium used as starter is 等人，2007）。

型的乳酸菌作为发酵剂
one of the main factors that influences the synthesis of bioactive 一个主要的影响因素，合成的生物活性
peptides in dairy products (Gobbetti et al., 2002). Indeed, it is
多肽乳制品（gobbetti等人。

，2002）。

事实上，它是
well known that lactic acid bacteria differ as for proteolytic
众所周知，乳酸菌差异蛋白
activity. In vivo studies showed that ACE-inhibitory peptides are 活动。

体内研究表明，ACE抑制肽
moderately hypotensive and foods containing them may be
中度低血压和食品含有这些可能是
considered as co-adjuvant in the treatment of mild hypertension 视为共同辅助对轻度高血压的治疗
(Seppo et al., 2003). Moreover, it has been shown that lactic acid （塞伯等人。

，2003）。

此外，它已经表明，乳酸
bacteria have the capacity to synthesize g-aminobutyric acid
细菌有能力合成g -氨基丁酸
(GABA) from L-glutamate through glutamate decarboxylase
（氨基丁酸）从L -谷氨酸通过谷氨酸脱羧酶
activity. During milk fermentation a high level of L-glutamate
活动。

牛奶发酵高水平的L -谷氨酸
may be theoretically liberated since native caseins contain a high 可能是理论上的解放自本土酪蛋白含有高
proportion of this amino acid. GABA, a non-protein amino acid,
这个比例的氨基酸。

丁酸，一种非蛋白质氨基酸，
possesses well known physiological functions such as neurotransmission, 具有众所周知的生理功能，如神经传递，
induction of hypotension, and diuretic and tranquilizers
诱导性低血压，和利尿剂和镇静剂
effects (Jakobs et al., 1993; Guin Ting Wong et al., 2003).
影响（雅科布斯等人。

，1993；因皇廷等人。

，2003）。

This paper reports on investigations aimed at developing and
本文报告的调查，旨在开发和
characterizing a fermented goats’ milk enriched of GABA and ACEinhibitory 表征发酵山羊'牛奶富含γ-氨基丁酸和aceinhibitory
peptides through the use of selected multiple starter
肽通过使用选定的多个启动器
cultures.
文化。

2. Materials and methods
2。

材料与方法
2.1. Raw milk, microorganisms and ingredients
2.1。

原料乳，微生物和成分
Milk samples were collected when goats (Ionica breed) were 9–
牛奶样品收集当山羊（ionica品种）9–
11 weeks in lactation and analysed for chemical composition.
11周的哺乳期和分析化学成分。

Protein content was determined by the Kjeldahl method (AOAC,
蛋白质含量测定凯氏定氮法（原，
2003a) using a VELP DK6 heating digester and a VELP UDK 130 2003年）使用维尔普dk6加热蒸煮和维尔普虚幻引擎开发包130 distillation system (VELP Scientifica, Usmate Milan, Italy). Fat was
蒸馏系统（维尔普scientificaUs mate，米兰，意大利）。

脂肪的determined by the Gerber method, according to Stelios and
确定的方法，根据斯泰利奥斯和
Emmanuel (2004). Lactose was determined by an enzymatic
艾曼纽（2004）。

乳糖是所确定的酶
method, using the Megazyme (Megazyme International Ireland
方法，使用megazyme（megazyme国际爱尔兰
Ltd., Bray, Ireland) kit K-LACGAR. Ashes were determined by dry
公司，布雷，爱尔兰）k-lacgar套件。

灰烬测定干
ashing the samples in a muffle furnace at 550 C (AOAC, 2003b).
灰化样品在马弗炉在550摄氏（化学家，2003年）。

The pH, determined by a pH meter (Beckmann F40, Beckmann, RIIC 值，确定一个值（贝克曼F，贝克曼，riic
Ltd, High Wycombe, UK) was ca. 6.55 and titratable acidity, determined 有限公司，海威科姆，英国）约为6.55，可滴定酸，确定
according to the AOAC Official Method no. 920.124 (AOAC,
根据官方920.124号（原测定方法，
2003c), was 0.125% (g of lactic acid/100 g of milk). Goats were
软件），0.125%（克乳酸/100克牛奶）。

山羊
grazed on pasture and milking was carried out twice daily, manually.
在牧场放牧、挤奶进行每天两次，手动。

After milking, the milk was immediately refrigerated to ca. 4 C
后挤奶，牛奶是立即冷藏约4丙
and within 3 h stored in a freezer (Ignis ICF410, Whirlpool, Siena,
3小时内和储存在冰箱（1icf410，惠而浦，锡耶纳，
Italy) at 20 C until used.
意大利）在20直到使用。

Lactic acid bacteria (S. thermophilus CR12, Lactobacillus casei
嗜热乳酸菌（Cr 12，干酪乳杆菌
LC01, Lactobacillus helveticus PR4, L. acidophilus 2949 and Lactobacillus lc01pr4，瑞士乳杆菌，嗜酸乳杆菌2949和乳酸菌
plantarum 1288) belonging to the Culture Collection of the
1288）属文化的收集
Department of Plant Protection and Applied Microbiology,
植物保护系和应用微生物学，
University of Bari, were routinely propagated in M17 (Oxoid Ltd,
巴里大学，经常被传播（蛋白多有限公司，
Basingstoke, Hampshire, UK) (streptococci) or MRS (Oxoid Ltd,
贝辛斯托克，汉普郡，英国）（链球菌）或夫人（蛋白有限公司，Basingstoke, Hampshire, UK) broth (lactobacilli) at 30 C (L. casei
贝辛斯托克，汉普郡，英国）肉汤（乳酸菌）在30℃（L干干酪
LC01 and L. plantarum 1288) or 37 C (S. thermophilus CR12, L.
lc01和植物乳杆菌1288）或37℃（嗜热链球菌Cr 12，L .
helveticus PR4 and L. acidophilus 2949).
瑞士pr4和嗜酸乳杆菌2949）。

Sodium caseinate was produced from goats’ milk as described
酪蛋白酸钠生产的山羊'牛奶的描述
by Minervini et al. (2003). The pectin preparation was provided by
由梅诺菲尼等人。

（2003）。

果胶的制备提供了
Lippolis srl (Putignano, Bari, Italy) and contained ca. 115 g kg 1
黎波里斯里亚尔（PUTIGNA NO，巴里，意大利）和包含约115克公斤1 moisture and 770 g kg 1 pectin; the degree of esterification for
水分和770克公斤1果胶的酯化度为；
pectin was ca. 63% (HMP). Threonine was purchased from Sigma
果胶是约63%（公司）。

苏氨酸购买西格玛
Chemical Co. (Milan, Italy).
化工有限公司（米兰，意大利）。

2.2. Manufacture of fermented milks
2.2。

生产发酵乳
Taking into account that it is difficult to manufacture a fermented
考虑到这是难以制造发酵
goats’ milk endowed with a good consistency, at the beginning of
山羊奶具有很好的一致性，在开始
the experimental work some trials were carried out with the aim to
实验工作进行了一些试验的目的
set a protocol for manufacturing a product with a good consistency
建立一个协议制造的产品具有很好的一致性
coagulum. All the fermented milks (FMs) were manufactured
凝结物。

所有的发酵奶（柔性制造系统）制造
according to the following steps: addition of sodium caseinate
按照下列步骤：添加酪蛋白酸钠
(15.0 g kg 1) and pectin (2.5 g kg 1) to improve coagulum consistency, （15克公斤1）和果胶（2.5克公斤1）提高凝固的一致性，
and of threonine (0.8 g kg 1) to increase the concentration of
和苏氨酸（0.8克公斤1）增加浓度
acetaldehyde; heat treatment (70 C for 30 min, while agitating) to
乙醛；热处理（70为30分钟，而搅拌）对
inactivate non-spore-forming pathogenic and spoilage microorganisms, 灭活孢子形成的致病微生物和腐败，
and to improve the distribution of the ingredients; rapid
和改善分配的成分；快速
cooling to 40 C and inoculation with a single- or mixed-starter
冷却到40摄氏接种单-或mixed-starter
culture, as indicated in Table 1. In details, heat treatment of milkwas
文化，如表1。

在细节上，热处理milkwas
conducted in a bain-marie, while milk temperature was monitored
在水浴，而牛奶温度监测
by inserting a penetration probe of a Dualtemp thermometer (TFA
插入一个贯穿探针的dualtemp温度计（三氟
Dostmann Ltd, Reicholzheim, Germany). The time needed for the
dostmann有限公司，reicholzheim，德国）。

所需的时间
center of a 500 ml milk bulk to reach 70 C was ca. 10 min. Subsequently, 中心的500毫升牛奶体积达到70摄氏约10分钟。

随后，
heat-treated milk was rapidly cooled (ca. 5 min) to 40 C in
热处理牛奶是迅速冷却（约5分钟）40丙型
a ice-bath. Starter lactic acid bacteria were prepared as follows: 24 h
一个冰浴。

乳酸菌发酵制备如下：24小时
old cells cultured inM17 or MRS broth were used to inoculate (1%, v/
老细胞培养inm17或肉汤被用来接种（1%，5 /
v) reconstituted 10% (w/w) skim milk from Oxoid Ltd. (Basingstoke,
五）重组10%（瓦特/瓦）脱脂乳蛋白（从公司网站，
Hampshire, UK). Cell growth in reconstituted skim milk was
汉普郡，英国）。

细胞生长在改组脱脂牛奶
determined by measuring the optical density of the culture at
通过测量光密度的文化
600 nm (Ultrospec 3000, Pharmacia Biotech, Uppsala, Sweden)
600纳米（ultrospec3000，溴酚蓝，乌普萨拉，瑞典）
after clarification by addition of sodium citrate (Exterkate, 1984).
澄清后，通过添加柠檬酸钠（exterkate，1984）。

One single preculture for each starter was prepared and used, even
一个单一的预为每个启动准备和使用，即使
for mixed started fermented milks. Twelve h-cultured skim milk
混合开始发酵奶。

十二h-cultured脱脂牛奶
was used to inoculate (1%, v/v) goats’ milk to obtain approximately
被用来接种（1%，体积比）山羊'牛奶获得约
7.0 log cfu g 1. Inoculated milkwas stirred, bottled and incubated at
7日志祖1克。

接种milkwas搅拌，瓶装和培养在
30 or 37 C, depending on the optimal temperature of the lactic acid
30个或37，取决于最佳温度的乳酸
bacteria inoculated (Table 1), until pH 4.6 was reached. Finally, FMs
细菌接种（表1），直至达到4.6。

最后，柔性制造系统
were cooled to 4–6 C and stored at 4 C.
被冷却到4–6和储存在4C
Three ba tches of goats’ milk were collected on different days and
三批山羊'牛奶收集不同天和
on each occasion were used for the manufacture of the eight types
每一次被用于制造的八种类型
of FM.
调频。

Table 1
表1
Lactic acid bacteria used as starters, incubation temperature and time needed to
乳酸菌作为起动器，孵化温度和所需的时间
reach pH 4.6 for different fermented goats’ milks.
达到4.6不同发酵山羊奶
Lactic acid bacteria Incubation
乳酸菌培养
temperature
温度
( C)
（丙）
Time
时间
(h)
（小时）
FM1 Streptococcus thermophilus CR12 简化嗜热链球菌Cr 12
Lactobacillus casei LC01
干酪乳杆菌lc01
Lactobacillus helveticus PR4
瑞士乳杆菌pr4
37 3.5
373.5
FM2 S. thermophilus CR12
fm2嗜热型
L. casei LC01
Llc01干酪
Lactobacillus plantarum 1288
植物乳杆菌1288
37 3.5
373.5
FM3 S. thermophilus CR12
fm3嗜热型
L. casei LC01
Llc01干酪
Lactobacillus acidophilus 2949
嗜酸乳杆菌2949
37 3.5
373.5
FM4 S. thermophilus CR12 37 4.5
fm4嗜热链球菌4.5型37
FM5 L. casei LC01 30 7.5
fm5干酪lc01307.5L。

FM6 L. helveticus PR4 37 13
fm6属瑞士pr43713
FM7 L. plantarum 1288 30 8
fm7植物乳杆菌1288308
FM8 L. acidophilus 2949 37 5
fm8嗜酸乳杆菌2949375
2.3. Microbiological analyses and determination of pH
2.3。

微生物分析和PH值的测定
Numbers of total aerobic mesophilic bacteria, total enterobacteria, 总人数中温好氧菌，总肠杆菌，
moulds and yeasts were determined on heated milk by
霉菌和酵母菌进行加热的牛奶
plating on Plate Count Agar, Violet Red Bile Glucose Agar, and Malt
电镀在平板计数琼脂，紫红胆盐葡萄糖琼脂，和麦芽
Agar medium, respectively. All media were purchased from Oxoid
琼脂培养基，分别。

所有的媒体购买来自奶牛
(Oxoid Ltd, Basingstoke, Hampshire, UK). Plates of Plate Count Agar （蛋白有限公司，贝辛斯托克，汉普郡，英国）。

平板计数琼脂平板
and Malt Agar were incubated at 30 C for 72 h, whereas plates of
麦芽琼脂培养在30摄氏为72小时，而板
Violet Red Bile Glucose Agar were incubated at 37 C for 48 h.
紫红胆盐葡萄糖琼脂培养在37摄氏48小时
The number of lactic acid bacteria was estimated at the end of
乳酸菌的数量估计在结束
milk fermentation, and after 45 days of storage at 4 C, by plating
牛奶发酵，经过45天的存储在4摄氏，电镀
on MRS (for lactobacilli) and M17 (for streptococci) agar media
夫人（乳杆菌）和多（链球菌）琼脂培养基
(Oxoid Ltd, Basingstoke, Hampshire, UK). In case of FMs produced
（蛋白有限公司，贝辛斯托克，汉普郡，英国）。

在柔性制造系统生产情况with both thermophilic and mesophilic lactobacilli (e.g. FM1), MRS
与高温和中温乳酸菌（如FM 1），夫人
plates were incubated for 48 h at 45 C or at 30 C, in order to
板培养48小时，45℃或30℃，为
evaluate counts of thermophilic and mesophilic lactobacilli,
评价和中温嗜热乳酸杆菌计数，
respectively. In case of FM2, that contained, besides S. thermophilus, 分别为。

在案件fm2，包含，除了嗜热链球菌，
two mesophilic lactobacilli (L. casei and L. plantarum), colonies
2嗜温乳酸菌（L .干酪乳杆菌和植物乳杆菌），殖民地
picked up from MRS plates incubated at 30 C for 48 h were subjected 拾起太太板孵育30丙48小时受到
to DNA extraction. In details, a single colony was suspended
去氧核糖核酸的提取。

在细节，一个单一的殖民地被暂停
in 20 mL of microLYSIS buffer (Labogen, Terrazzano di Rho, Italy) and 在20毫升的microlysis缓冲（labogen，terrazzano迪卢，意大利），subjected to a heating–cooling treatment (5 min at 65 C, 2 min at
受到加热–冷却（5分钟，65，2分钟
96 C, 4 min at 65 C, 1 min at 96 C, 1 min at 65 C, and 30 s at 96，4分钟在65，1分钟在96，1分钟在65丙，和30时
96 C) using a thermal cycler (GeneAmp PCR System 9700: PE
96丙）使用热循环仪（geneamp聚合酶链反应系统9700：体育Applied Biosystems, Norwalk, CT), as indicated by the manufacturer. 美国应用生物系统公司，诺沃克，康涅狄格州），如制造商。

Subsequently, the DNA region codifying for rRNA 16S was
随后的基因区域，基因研究是编纂
amplified through polymerase chain reaction (PCR), using the
扩增通过聚合酶链反应（聚合酶链反应），使用
primer pair LpigF/LpigR (De Angelis et al., 2006), in order to allot
引物对lpigf/lpigr（少校等人。

，2006），以便分配
each single colony to a certain species. PCR and subsequent gel
每一个单菌落进行某些物种。

聚合酶链反应和随后的凝胶electrophoresis conditions, sequences determination and comparison 电泳条件，序列测定与比较
in disposable databases were as described by de Candia et al.
一次性使用的数据库进行描述的克里特岛等。

(2007).
（2007）。

The values of pH at the beginning, at the end of fermentation,
值的值在一开始，结束时发酵，
and after 45 days of storage at 4 C were determined by using
经过45天的存储在4摄氏测定
a Foodtrode pH electrode (Hamilton Bonaduz AG, Switzerland).
一个foodtrode电极（汉密尔顿博纳图斯股份公司，瑞士）。

2.4. Determination of carbohydrates and
2.4。

测定碳水化合物和
fermentation end-products
发酵终产物
Lactose, galactose, glucose, D- and L-lactic acid were determined
乳糖，半乳糖，葡萄糖，-和L -乳酸测定
by enzymatic methods, using the Megazyme (Megazyme International 酶解方法，使用megazyme（megazyme国际
Ireland Ltd., Bray, Ireland) kits K-LACGAR (lactose and
爱尔兰公司，布雷，爱尔兰）包k-lacgar（乳糖，
galactose), D-Glucose HK (glucose), and K-DLATE (D- and L-lactic 半乳糖），葡萄糖（葡萄糖），香港和k-dlate（四-、L
acid). Acetaldehyde was determined by an enzymatic method (Kit. 酸）。

乙醛测定酶法（试剂盒。

Art. No. WCP647 CL-10 Plus, Diffchamb Italy S.r.l.. San Giuliano
艺术。

第wcp647cl-10加，diffchamb意大利公司。

圣格里诺Milanese, Italy).
米兰，意大利）。

2.5. Determination of ACE-inhibitory activity
2.5。

测定ACE抑制活性
The pH 4.6-soluble fractions of FMs, obtained through centrifugation 值4.6-soluble分数，通过离心
(10,000 g, 10 min, 4 C), were assayed for ACE-inhibitory （10000克，10分钟，4丙），检测ACE抑制
activity by the method reported by Nakamura et al. (1995) with
活动的方法报告的中村等人。

（1995）与
some modifications. One hundred mL of a 5 mM solution of hippuryl- 一些修改。

一百毫升的5毫米解决hippuryl—
L-histidil-L-leucine in 0.1 M sodium borate buffer containing
l-histidil-l-leucine在0.1米的硼酸钠缓冲液含有
0.3 M NaCl was mixed with 30 mL of pH 4.6-soluble fraction, or
0.3米氯化钠混合30毫升的PH值4.6-soluble分数，或
water, and 20 mL of ACE (100 mU mL 1) from rabbit lung; the
水，和20毫升的王牌（100亩毫升兔肺的1）；
mixture was incubated for 60 min at 37 C and the reaction was
混合培养60分钟在37和反应
stopped with 125 mL of HCl 1N. Controls without ACE added were
停止与125毫升的盐酸和不加控制的王牌。

included for each measurement. The hippuric acid liberated by ACE
包括为每个测量。

马尿酸解放的王牌
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was extracted with 0.85 mL of pure ethyl acetate and, after removal
提取0.85毫升纯乙酸乙酯，去除后
of ethyl acetate by vacuum evaporation, dissolved in 2 mL of
乙酸乙酯的真空蒸发，溶解在2毫升
distilled water and determined spectrophotometrically at 228 nm.
蒸馏水和确定分光在228纳米。

The percentage of ACE-inhibitory activitywas calculated as follows: ACEIactivitywas百分比计算如下：
(B A)/(B C) 100, where A ¼ absorbance in the presence of
（二一）/（二丙）100，在¼吸光度的存在
both ACE and pH 4.6-soluble fraction, B ¼ absorbance without pH
血管紧张素转换酶和4.6-soluble分数，乙¼吸光度值无
4.6-soluble fraction, and C ¼ absorbance without ACE.
4.6-soluble分数，和¼吸光度无王牌。

2.6. Determination of free amino acids and g-aminobutyric acid
2.6。

测定游离氨基酸和g -氨基丁酸
Total and individual free amino acids (FAAs) and g-aminobutyric
和个人总游离氨基酸（原子吸收光谱法）和g-aminobutyric
acid contained in the pH 4.6-soluble nitrogen fractions of FMs were 含酸PH值4.6-soluble氮组分的柔性制造系统
analyzed by a Biochrom 30 series Amino Acid Analyser (Biochrom 分析了biochrom30系列氨基酸分析仪（biochrom
Ltd., Cambridge Science Park, UK) with a sodium-cation-exchange 有限公司，剑桥科学园区，英国）与sodium-cation-exchange column (20 by 0.46 cm inner diameter). A mixture of amino acids at 柱（20的内径0.46厘米）。

混合氨基酸
known concentration (Sigma Chemical Co., Milan, Italy) was added 已知浓度（西格玛化工有限公司，米兰，意大利）被添加
with cysteic acid, methionine sulphoxide, methionine sulphone,
与半胱氨酸，蛋氨酸亚砜，蛋氨酸砜，
tryptophan, ornithine, taurine, hydroxy-proline and g-aminobutyric 色氨酸，牛磺酸，鸟氨酸，羟脯氨酸和g-aminobutyric
acid (GABA), and used as standard. Proteins and peptides in
酸（γ-氨基丁酸），和使用的标准。

蛋白质和多肽
the samples were precipitated by addition of 5% (v/v) cold solid
对样品进行了沉淀除5%（体积比）冷固
sulphosalicylic acid, holding at 4 C for 1 h and centrifuging at
磺基水杨酸，保持在4摄氏和离心在1小时
15,000 g for 15 min. The supernatant was filtered through
15000克15分钟过滤上清液通过
a 0.22 mmpore size filter and diluted, when necessary, with sodium
0.22mmpore大小的过滤和稀释，必要时，钠
citrate (0.2 M, pH 2.2) loading buffer. Amino acids were postcolumn 柠檬酸（0.2米，2.2）缓冲液。

氨基酸的柱
derivatised with ninhydrin reagent and detected by
衍生化与茚三酮试剂和检测
absorbance at 440 (proline and hydroxy-proline) or 570 nm (all the 吸光度在440（脯氨酸和羟脯氨酸）或570纳米（所有
other amino acids).
其他氨基酸）。

2.7. Statistical analyses
2.7。

统计分析
For each FM, three batches were analyzed in triplicate, and
每个调频，三批进行了分析，一式三份，和
experimental data were subjected to analysis of variance (ANOVA) 实验数据进行了方差分析（方差分析）
and pair-comparison of treatment means was achieved using
和副比较治疗手段取得使用
Tukey’s procedure at P < 0.01, using a statistical software Statistica 图基的程序在<0.01，使用统计软件统计
for Windows (Statsoft, 1995).
窗口（statsoft，1995）。

3. Results and discussion
3。

结果与讨论
3.1. Selection of starter cultures based on production of
3.1。

选用起动机文化基础上的生产
ACE-inhibitory peptides and GABA
ACE抑制肽和γ-氨基丁酸
The goats’ milk used had the following chemical composition:
山羊奶使用了以下的化学成分：
protein 33.0 0.4 g kg 1, fat 46.5 0.9 g kg 1, lactose 48.2 0.2 g 蛋白330.4克公斤1，脂肪46.50.9克公斤1，乳糖48.20.2克
kg 1; ash 7.0 0.5 g kg 1.
1公斤；灰分70.5克公斤1。

No yeasts, moulds, neither enterobacteria were found in goats’
没有酵母，霉菌，既不肠杆菌被发现在山羊
milk after heat treatment, whereas a count of total aerobic mesophilic 牛奶经过热处理，而计数总好氧嗜温
bacteria was 2.1 0.3 log cfu g 1. After sodium caseinate,
细菌是2.10.3日志祖1克。

在酪蛋白酸钠，
pectin and threonine had been added to milk and after milk had
果胶和苏氨酸被添加到牛奶和奶后有
been inoculated with starter lactic acid bacteria, the initial pH was
接种乳酸菌发酵，初始值为
6.10. Five different lactic acid bacteria, used alone or in combination 6.10。

五种不同的乳酸菌，单独使用或组合
to ferment goats’ milk, were screened for healthy properties. The
发酵山羊奶，筛选健康特性。

本
ACE-inhibitory activity and the concentration of GABA of the FMs
ACE抑制活性和浓度的丁酸的柔性制造系统
(FM1–8) are shown in Table 2. The highest percentage (P < 0.01) of （FM 1–8）表2所示。

最高比例（<0.01）的
ACE-inhibition (82.0%) was found in the pH 4.6-soluble fraction of
血管紧张素转换酶抑制（82%）被发现在4.6-soluble分数的值
goats’ milk fermented with S. thermophilus CR12, L. casei LC01 and L. 山羊'牛奶发酵嗜热链球菌L型，lc01和干酪
helveticus PR4 (FM1). Except for FM manufactured with L. acidophilus 瑞士pr4（FM 1）。

除了调频用嗜酸乳杆菌
(FM8), all the other FMs had percentage of ACE-inhibitory
（fm8），所有其他的柔性制造系统有比例的ACE抑制
activity between 34.3 and 51.3%. As previously shown (Gobbetti
活性之间的34.3和51.3%。

如前所示（gobbetti
et al., 2004), the ACE-inhibitory activity of the pH 4.6-soluble
等人。

，2004），其ACE抑制活性的4.6-soluble
fraction of fermented milks may be due to bioactive hypotensive
部分发酵奶的原因可能是生物活性降压
peptides generated during milk fermentation, thanks to the
多肽产生的发酵奶，多亏
proteolytic activity of lactic acid bacteria. It has to be stresses that,
蛋白活性乳酸菌。

它已被强调，
the capacity of lactic acid bacteria to generate ACE-inhibitory
能力的乳酸菌产生ACE抑制
peptides during milk fermentation is strain-dependent, as previously
肽发酵奶是应变依赖，如以前
reported by Fuglsang et al. (2003). The renowned degree of
报告的fuglsang等人。

（2003）。

著名的
casein heterogeneity across milk of different species, in terms of
牛奶酪蛋白的异质性在不同的物种，在条款
relative proportion of caseins, primary structure and native
相对比例酪蛋白，主要结构和本地
conformation of individual caseins (Ng-Kwai Hang and Grosclaude,
构象的个人酪蛋白（ng-kwai挂grosclaude，
1992), may influence the susceptibility of caseins to enzymatic
1992），可能影响的敏感性酪蛋白酶
hydrolysis and to generation of bioactive peptides, as previously
水解，生成活性肽，如以前
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Table 2
表2
Percentage of ACE inhibition and concentration of g-aminobutyric acid in fermented
血管紧张素转换酶的抑制率和浓度的g -氨基丁酸发酵
goats’ milks manufactured with different lactic acid bacteria; n ¼ 9.
山羊奶的生产具有不同的乳酸菌的¼；9。