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Wei Chen Editor
Lactic Acid
Bacteria
Bioengineering and Industrial
Applications
Lactic Acid Bacteria
Wei Chen
Editor
Lactic Acid Bacteria

Bioengineering and Industrial Applications
Editor
Wei Chen
Jiangnan University
Wuxi, China
The print edition is not for sale in The Mainland of China. Customers from The Mainland of
China please order the print book from: Science Press.
ISBN 978-981-13-7282-7 ISBN 978-981-13-7283-4 (eBook)

https://doi.org/10.1007/978-981-13-7283-4
© Springer Nature Singapore Pte Ltd. and Science Press 2019
This work is subject to copyright. All rights are reserved by the Publishers, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publishers, the authors, and the editors are safe to assume that the advice and information in this
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or the editors give a warranty, express or implied, with respect to the material contained herein or for any
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claims in published maps and institutional affiliations.
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The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore
Contents
1 Lactic Acid Bacteria and γ-Aminobutyric Acid and Diacetyl�� 1

Shunhe Wang, Pei Chen, and Hui Dang
2 Lactic Acid Bacteria and Conjugated Fatty Acids�� 21
Wei Chen, Bo Yang, and Jianxin Zhao
3 Lactic Acid Bacteria and B Vitamins �� 43
Wanqiang Wu and Baixi Zhang
4 Lactic Acid Bacteria and Bacteriocins �� 61
Qiuxiang Zhang
5 Lactic Acid Bacteria Starter �� 93
Wei Chen and Feng Hang
6 Lactic Acid Bacteria and Fermented Cereals�� 145
Bowen Yan and Hao Zhang
7 Lactic Acid Bacteria and Fermented Fruits and Vegetables�� 181
Bingyong Mao and Shuang Yan
8 Lactic Acid Bacteria and Fermented Meat Products�� 211
Shumao Cui and Zhexin Fan
9 The Preparation Technology of Pharmaceutical
Preparations of Lactic Acid Bacteria �� 227
Wei Chen and Linlin Wang
10 Lactic Acid Bacteria in Animal Breeding and Aquaculture�� 257
Gang Wang and Xing Jin
v
Chapter 1
Lactic Acid Bacteria and γ-Aminobutyric
Acid and Diacetyl
Shunhe Wang, Pei Chen, and Hui Dang
1.1 Lactic Acid Bacteria and γ-Aminobutyric Acid
1.1.1 Introduction of γ-Aminobutyric Acid

1.1.1.1 γ-Aminobutyric Acid
γ-Aminobutyric acid (GABA), or γ-ammonia butyric acid, is a kind of nonprotein

amino acid which widely exists in vegetables and animals. It exists in the seeds,
roots, and tissue fluid of many plants, such as Glycine L., Panax, herbal, and in ani-
mals almost exclusively present in nervous tissues. The content of GABA in brain
tissue is 0.1~0.6mg/g, and immunology research show that substantia nigra contain
the highest concentration of GABA (Krajnc et al. 1996). Meanwhile GABA is also
present in microorganisms, such as yeast, Lactobacillus, and Escherichia coli.
GABA was discovered in the metabolite of plants and microorganisms in 1883.
Awapara et al. (1950), Roberts and Frankel (1950), and Udenfriend (1950) found
that the content of GABA was relatively high in the brain of mammals, but the bio-
logical significance was unknown. Roberts reported that GABA was a product of
glutamate through α-decarboxylation. Krnjević and Schwartz (1966) researched
that GABA played a part in the brain of mammals as an endogenous neurotrans-
mitter and GABA was confirmed as an inhibitory neurotransmitter in the Second
International Symposium on GABA in 1975.
S. Wang (*)
Jiangnan University, Wuxi, China
e-mail: wangshunhe@jiangnan.edu.cn
P. Chen
Shaanxi Radio & TV University, Xi’an, China
H. Dang
Shaanxi Normal University, Xi’an, China
© Springer Nature Singapore Pte Ltd. and Science Press 2019 1

W. Chen (ed.), Lactic Acid Bacteria,
https://doi.org/10.1007/978-981-13-7283-4_1
2 S. Wang et al.
Many studies in recent years have reported the physiological function of GABA,
including delaying senescence of nerve cells, lowering blood pressure, repairing the
skin, treating mental illnesses, and regulating cardiac arrhythmia and hormone
secretion (He Xipu et al. 2007). Scientists from all around the world studied the
synthesis technology and its related product. GABA could be produced by chemical
synthesis, plant enrichment, and microbiological fermentation. Sawai et al. (2001)
researched that the content of GABA in tea could be enhanced through a variable
anaerobic-aerobic process. Meanwhile, it also could be produced by controlling the
germination condition of brown rice and rice germ. Xia Jiang et al. reported that
Lactobacillus brevis CGMCCNO.1306 was isolated from unpasteurized raw milk
and the capacity of GABA was 76.36 g/L after the mutation breeding (Xia Jiang
2006; Xia Jiang et al. 2006).
1.1.1.2 General Physicochemical Property
The chemical formula is C4H9NO2 and molar mass is 103.120 g/mol. It appears as a
white microcrystalline powder, easily soluble in water, slightly soluble in ethanol,
and insoluble in cold ethanol, benzene, and ether. The dissociation constant of
pKCOOH and pKNH3 were 4.03 and 10.56, respectively. GABA has no optical
rotation, the melting point was 203~204°C. Its products of decomposition were pyr-
rolidone and water (Chen Lilong and Shi 2010). In most part, GABA exists in the
form of amidogen with positive charge or carboxyl with negative charge. The state
of GABA determined its molecular conformation. In the gaseous state, the molecu-
lar conformation is highly folded due to the electrostatic interaction of two charged
groups; in solid state, the molecular conformation is extended due to the intermo-
lecular interaction between the two groups. When in liquid state, molecular confor-
mations exist in both states. The various conformations of GABA are combined
with different receptor proteins, which play significant physiological functions.
1.1.2 GABA Production of Lactic Acid Bacteria
The production of GABA by microbial fermentation is to use the glutamate decar-

boxylase in organisms as a catalyst, which GABA is produced from L-glutamate
or its sodium salt by α-decarboxylation reaction. Glutamate decarboxylase is the
single enzyme to generate GABA from L-glutamate or its sodium salt, which was
discovered in bacteria, archaea, and eucaryotic microbes. Compared with chemical
synthesis and plant enrichment, GABA from microorganism fermentation, which
has low cost and high yield, could be used in food safely.
Lactic acid bacteria, as a safe microorganism (GRAS), are widely used in foods,
such as yogurt, cheese, and pickles. Many studies show that lactic acid bacteria have
glutamate decarboxylase which generates GABA from L-glutamate by decarboxyl-
1 Lactic Acid Bacteria and γ-Aminobutyric Acid and Diacetyl 3
ation reaction. Nomura et al. (1998) reported that the content of GABA was 383 mg/
kg in cheese, in which a strain of Lactococcus lactis 01–7 with high GABA was
isolated. Komatsuzaki et al. (2005) researched that a strain of Lactobacillus paraca-
sei NFRI7415 with high GABA was isolated from traditional fermented food and
the capacity of GABA was 302 mmol/L. Xu Jianjun et al. (2002) isolated a strain of
Lactococcus lactis with high GABA for the first time interiorly, and the amount of
GABA in the fermentation broth was 250 mg/100 ml, which was fermented in 25 L
tank for 72 h. It could be used in the development of yogurt or as an ingredient in
other foods. Cui Xiaojun et al. (2005) also reported the screening and fermentation
conditions of high GABA-producing Lactobacillus, and the capacity of GABA was
5.4 g/L in the fermentation broth. Xia Jiang et al. (2006) isolated a strain of
Lactococcus lactis hjxi-01, and the maximum accumulation concentration of GABA
was 7 g/L in the medium containing 5% L-sodium glutamate (Xia Jiang 2006). And
on this basis, the initial strain was mutagenized by ultraviolet rays and radiation.
Finally, a high-yield mutant hjxj-80,119 was obtained, which was passed on for 12
consecutive generations and had stable genetic characters. Compared with the origi-
nal strain hjxj-01, the average yield increased by 142.9%, and the average product
was 17 g/L. Using neural network and particle swarm optimization algorithm, the
researchers optimized the fermentation conditions of lactic acid bacteria in the
shake flask, and the accumulated amounts of GABA was 33.4 g/L. After the optimi-
zation of indirect fermentation conditions, the concentration of GABA reached
107.5 g/L. Siragusa et al. (2007) isolated 12 different species of lactic acid bacteria
with high GABA from 22 kinds of Italian cheese. Five species of Lactobacillus,
including Lactobacillus casei PF6, Lactobacillus bulgaricus PR1, Lactococcus lac-
tis PU1, Lactobacillus plantarum C48, and short lactic acid bacteria PM17, had
strong ability to synthesize GABA. And the result of PCR acquired the glutamate
decarboxylase gene. The homology was analyzed by amino acid analyzer with
85%~99%. Ji Linli et al. (2008) isolated nine strains of lactic acid bacteria with high
GABA from traditional fermented milk. The strains WH11–1 and M10–4-3 were
identified as Lactococcus lactis subsp. lactis through their traditional morphologi-
cal, physiological, biochemical, and chemical characteristics and 16S rDNA gene
identification. Fan et al. (2012) isolated Lactobacillus brevis CGMCC1306 with
high glutamate decarboxylase active from fresh and unpasteurized milk and further
analyzed the glutamate decarboxylase, which provided a basis for an approach that
could use glutamate decarboxylase and high-density culture to produce GABA in
the future. Xian Qianlong (2013) isolated 14 strains of lactic acid bacteria produc-
ing GABA, and two strains, including QL-14 (0.39 g/L) and QL-20 (0.584 g/L), had
relatively high GABA content. Through morphological features, physiological and
biochemical experiment, and 16S rDNA gene identification, strains of QL-14 and
QL-20 were identified as Lactobacillus plantarum and Lactobacillus casei,
respectively.
4 S. Wang et al.
1.1.3 Physiological Function of γ-Aminobutyric Acid
The current research about function of γ-aminobutyric acid mainly focus on the
treatment and improvement of the nervous system disease, high blood pressure,
liver function, and renal function, the impact on the physiology and reproduction,
and many other aspects.
1.1.3.1 Delaying the Senescence of Nerve Cells
Leventhal et al. (2003) researched that GABA could make the brain of aged macaque
in peak form, while as the growth of age, the brain GABA would reduce gradually.
Macaque visual function was similar with human, which would be deteriorated with
the increase of age. When brain in peak form, macaque could choose signal to
respond. Lack of GABA could lead to the degeneration of nerve cell in macaque,
and that would decline the ability of visual selection. Compared with younger
macaque, after injecting GABA and its agonist in an aging macaque, visual cortex
cells could be a greater degree to restore the ability of visual stimulus orientation
and directional selectivity, making the “senescence” cells show the characteristics
of being “young.”
Leventhal et al. (2003) studied the effects of electrophoretic application of
GABA by using multibarreled microelectrodes, and the agonist and antagonist of
GABA A-type receptor were used in individual V1 cells in old monkeys. Compared
with old monkeys, GABA A-type receptor antagonist played an important role in
neuronal responses in young monkeys, which indicated the degradation of GABA-
mediated inhibition with the increasing monkey age. On the other side, the treat-
ment of GABA resulted in improved visual function. The treated cells in area V1 of
old animals displayed the special characteristic of young cells.
1.1.3.2 Lower Blood Pressure
High blood pressure is a common human disease and a major risk factor for coro-
nary heart disease, stroke, or other cardiac-cerebral vascular diseases. According to
statistics, cardiac-cerebral vascular diseases, which were caused by high blood pres-
sure, caused about more than 12 million deaths yearly. Takahashi et al. (1959) first
reported the effect of GABA on the cardiovascular system, which found that GABA
had the strongest effect on cardiovascular activity in the γ-amino acid group. Later,
they also confirmed that 10 mg/kg GABA could reduce blood pressure and cause
bradycardia in rabbit, dog, and cat experiments. But its effect could be prevented by
resecting sympathetic nerve or blocking ganglion. In addition, this effect didn’t
need the vagus nerve, aortic nerve, and sinus nerve. Therefore, GABA might regu-
late blood pressure and heart rate by interacting central nervous system. Antonaccio
and Taylor (1977) injected 3~1000 g/kg GABA into the encephalocoele of anesthe-
tized cats, which decreased the blood pressure and heart rate with a dose-effect
relationship. It confirmed that GABA adjusted blood pressure and heart rate by
central nervous system for the first time. Kazami et al. (2002) also reported that
compound flavor containing GABA could significantly reduce the blood pressure of
patients with mild hypertension, which reduced systolic blood pressure by 6 mmHg
and diastolic blood pressure by 4 mmHg. The blood pressure of the normal group
remained unchanged, and no adverse reactions were observed. Hayakawa et al.
(2004) reported that GABA-enriched skim milk fermented by lactic acid bacteria
was fed to normal rat with spontaneous hypertension. The result showed that the
GABA-enriched milk had significantly lowered the blood pressure. Further study
found that the fermented milk didn’t contain antihypertensive peptides and the func-
tional component for lowering the blood pressure was GABA, which was similar to
the antihypertensive active ingredient in Astragalus membranaceus and manyinflo-
rescenced sweetvetch root.
1.1.3.3 Effects on Physiological Reproduction
The main inhibitory neurotransmitter in the brain was γ-aminobutyric acid. And it
could participate in the secretion regulation of anterior pituitary hormone at the
level of the hypothalamus or pituitary, which indirectly affected the function of the
ovary, oviduct, male gonads, and accessory organs and was closely related to the
movement of sperm and the production of steroid hormones. Murashima and Kato
(1986) found that the capacity of GABA in the tubal mucosa of rats was ten times
higher than that in the brain, and the concentration of GABA decreased in a gradient
from the oviduct fimbria to the connection of uterine and oviduct. Meanwhile,
research also showed that GABA is present in the male gonads and accessory
organs. GABA receptor was found on the surface of sperm membrane, suggesting
that GABA was related to sperm function. Roldan et al. (1994) reported that GABA
could significantly induce acrosome reaction in capacitated spermatozoa of mice
and human and had significant dose-effect relationship. Progesterone had promot-
ing effect to the reaction. Bian Shuling et al. (2002) reported that GABA could
increase the sperm acrosin activity and significantly increased Na+−k+-ATPase and
superoxide dismutase activity in normal and positive antisperm antibody (AsAb)
men and found that GABA could increase the sperm acrosin activity and signifi-
cantly increased Na+/K+-ATPase and superoxide dismutase activity, so it has impor-
tant theoretical significance and broad application outlooks in reproductive
physiology.
1.1.3.4 Treatment of Mental Illness
GABA was the most important inhibitory neurotransmitter in the central nervous
system of mammal, shellfish and certain parasitic worms and mediated over 40% of
inhibitory nerve conduction. The abnormality of GABA induced various nervous
system diseases, including excitotoxic reaction, epilepsy, insomnia, etc. (Mombereau
6 S. Wang et al.
et al. 2004). Since Tower first proposed that the occurrence of epilepsy was related
to GABA in the brain in the early 1960s, a large number of studies had shown that
the content of GABA in the spinal fluid of patients with epilepsy was significantly
lower than that of normal people and the extent of the decline was related to the type
of seizure. Many research showed that the content of GABA in the spinal fluid of
epileptic patients was significantly lower than that of normal people, and the decline
level was related with the type of epilepsy (Song Wei et al. 2008). GABA was a
special biochemical drug to treat intractable epilepsy. Experimental epilepsy could
be induced by GABA inhibitors, such as allylglycine, GABA A-type receptor, and
receptor antagonists, while GABA receptor agonist had anticonvulsive and antiepi-
leptic effects (Usuki et al. 2007). Okada et al. (2000) used GABA rice germ food
orally and found that it had the role of sedation, promoting sleep and resistance to
anxiety, and the total improvement rate of woman menopause syndrome and early
mental disorders was 75% in the elderly.
1 mmHg = 133.322 Pa
1.1.4 Application of γ-Aminobutyric Acid
The development of GABA-enriched foods began in 1986. GABA-enriched teas

were first successfully developed by Japan’s Ministry of Agriculture, Forestry, and
Fisheries in tea testing grounds for sale. In 1994, Japan’s Ministry of Agriculture,
Forestry, and Fisheries successfully developed GABA-enriched rice germs and rice
bran at China agricultural testing ground. Then, Japan developed the GABA-enriched
germinated brown rice and the healthy food materials with high GABA concentra-
tion which were fermented by Lactobacillus and yeast. In recent years, the research
and development of GABA-enriched healthy food, including plants (rice germ, rice
bran, green tea, pumpkin, etc.) and microorganisms (Lactobacillus, yeasts), have
become the research focuses at home and abroad (Cao Jiaxuan et al. 2008).
Lactobacillus starter is essentially used in fermenting milk, which ferments glu-
cose into lactic acid, providing the best environment for the formation of clots,
texture, and flavor. The anaerobic fermentation of Lactobacillus increased acidity,
which gave it the acid-resistant ability to maintain vitality at low pH, The low pH
environment maintained the activity of glutamate decarboxylase in Lactobacillus.
And glutamate decarboxylase consumed hydrion and reduced acidity, so that
Lactobacillus could produce the functional dairy products with GABA activity.
Nomura et al. (1998) reported that three of the seven commercial Lactobacillus
starters could produce GABA in skim milk culture and cheese-ripening process, in
which the yield was 383 mg/kg (Huang Yahui et al. 2005).
Fermented soybean food mostly used natural fermentation rich in microbial spe-
cies. Lactic acid bacteria widely existed in various kinds of fermented soybean
food. Lactic acid bacteria, producing GABA in fermented soybean food, improved
the flavor of soybean fermentation food, and the glutamic acid and its sodium salt
could be used to synthesize GABA, which provided a new idea for future research
on increasing GABA content and enhancing the function of soybean fermentation
food (Geng Jingzhang 2012).
1.2 Lactic Acid Bacteria and Diacetyl
1.2.1 Introduction of Diacetyl
Diacetyl, or butanedione (chemical name, 2,3-butanedione), is a yellow or light

green liquid with the molecular formula CH3COCOCH3. When diluted to 1 mg/L, it
has intensely buttery flavor and easily soluble in ethanol, ether, propylene glycol,
glycerol, and water (Guo Zheng 1998; Xie Haiyan and Yin Dulin 2000; Ai 1984).
Diacetyl has a relatively low flavor threshold (1.5~5.0 μg), so it can give the product
a strong flavor at a low concentration. Acetoin is the reductive form, and the fra-
grance is far inferior to diacetyl. Natural diacetyl existed in Foeniculum vulgare,
narcissus, tulip, raspberry, strawberry, lavender, citronella, rockrose, and cream
(Xie Haiyan and Yin Dulin 2000). In the food industry, as a flavor enhancer, diacetyl
was mainly used in soft drinks, cold drinks, baked food, candy, and other products.
In addition, diacetyl was also one of the taste and flavor compounds that play an
important role in fermented dairy products (Yang Lijie and Wang Junhu 2004). It
was also an important unpleasant odor source of beer and wine (Yang Lijie and
Wang Junhu 2004).
At present, Lactobacillus could be used alone or in combination with starter
culture to produce yogurt with the diacetyl as main flavor compounds in european
and american countries. Meanwhile, diacetyl was an essential flavor ingredient in
butter, yogurt, cream, cheese, and many nondairy products that require milk flavor.
And it was widely used in the preparation of flavoring butter essence, strawberry
essence, essence of suckling pig feed, and synthetic fats flavor enhancer and also
could be used as gelatin hardener and photographic adhesive. Although there are
many methods to synthesize diacetyl, it was only manufactured by few producers in
China. And because of the limitations of productive technology, the production of
diacetyl had low yield, poor quality, and high cost and had the difficulty in meeting
the requirements of replacing imported products. Diacetyl, which was used to pro-
duce essence and other products by perfumery plant, was imported from the
Netherlands, Japan, and other countries. And the import price was around 200 yuan/
kg. Domestic demand all year round was 30~40 t (Han Guangdian 1978; Wang
Zhen 1992).
In addition, diacetyl could inhibit the growth of many microorganisms, including
pathogenic bacteria and spoilage bacteria (Dan Tong and Zhang Heping 2013).
Trace amounts of diacetyl inhibited the growth of E. coli. And with lower system
pH, the bacteriostatic activity was strengthened (Jay et al. 1992), which indicated
8 S. Wang et al.
that lower pH enhanced the bacteriostatic activity of diacetyl (Meng Xiangchen

2009; Jay et al. 1992). On the contrary, glucose, acetate, and Tween-80 had an
antagonistic effect on the antibacterial activity of diacetyl (Meng Xiangchen 2009).
The reaction between diacetyl and arginine-binding protein interfered the use of
arginine by gram-negative bacteria and inhibited bacterial growth (Zheng Yingfu
et al. 2005).
1.2.2 Diacetyl Production of Lactic Acid Bacteria
Many microorganisms could produce diacetyl, such as Streptococcus, Leuconstoc,

Lactobacillus, Pediococcus, Oenococcus, and other microbial species (Bartowsky
and Henschke 2004). The leavening agents, including Lactobacillus bulgaricus and
Streptococcus thermophilus, were used to ferment milk in China. The two strains
had a symbiotic relationship and could promote the growth of each other in culture.
Generally, Streptococcus thermophilus was considered as the major strain to pro-
duce diacetyl and had larger contribution to the flavor of fermented milk (Meng
Xiangchen 2009). Scholars from the United States, the Netherlands, Denmark, and
other countries used genetic engineering and mutant breeding technology to select
and transform Lactobacillus with high diacetyl (Hua Chaoli and Zhao Zheng 2004).
However, it was still in the beginning in China. Song Huanlu (2002) studied the
production of diacetyl by Lactobacillus D21 and Lactococcus lactis NCIMB
8763 in milk and MRS medium. Yu Peng et al. (2006) used nitrosoguanidine to
mutate and breed the strain with high diacetyl. Liu Fang et al. (2006) conducted a
large-scale screening of Streptococcus thermophilus that produced high diacetyl.
Benson et al. (1996) overexpressed the coding genes for α-acetolactic acid synthe-
tase in α-acetolactic acid decarboxylase-defective Lactococcus and made the diace-
tyl production increased significantly. Boumerdassi et al. (1997) overexpressed the
coding genes for NADH oxidase (nox gene) in α-acetolactic acid decarboxylase-
defective Lactococcus. The production of diacetyl was increased by more than ten
times, and the growth of engineered bacteria remained unchanged.
1.2.3 roduction Way and Metabolic Regulation of Diacetyl

P
in Lactobacillus
1.2.3.1 Production Way of Diacetyl in Lactobacillus
1.2.3.1.1 Production of Diacetyl by Citric Acid Metabolic Pathways
Diacetyl is the key aroma of many fermented dairy products and also produces
unpleasant odor in wine and lemon juice (Jordan and Cogan 1988). The microorgan-
ism used to produce diacetyl was mainly Lactobacillus which could use citric acid.
Metabolism of citric acid by lactic acid bacteria was shown in Fig. 1.1. As was well
Fig. 1.1 Citric acid metabolic pathways in Lactobacillus
known, the two features of lactose fermentation and lactic acid production were
important to lactic acid bacteria in dairy products. And screening the strain that
could use citric acid to produce diacetyl was an important industry characteristic in
production of dairy products (Ma Guihua 1989; Borts 1963; Rodríguez et al. 2012).
Lactic acid bacteria which were able to take advantage of citric acid to produce
acetoin could be used to produce diacetyl. Therefore, lactic acid bacteria that pro-
duced diacetyl included Leuconostoc dextranicum, Leuconostoc citreum,
Streptococcus lactis, and some Bacterium lacticum (Yang Jiebin et al. 1996).
Citric acid was one of the most important substrates in the generation of diacetyl
by Lactobacillus. Citric acid was transferred into the cell by enzymes and then lysed
into acetic acid and oxaloacetic acid by lyase. Oxaloacetic acid was decomposed
into CO2 and pyruvic acid, while the process of pyruvate to diacetyl had not been
clearly explained (Hemme and Foucaud-Scheunemann 2004; Mcsweeney and
Sousa 2000). It is currently believed to be in two ways: The first was proposed by
Speckman and Collins (1968). It illustrated that active acetaldehyde was synthe-
sized from pyruvate and pyrithiamine, then condensed with acetyl-CoA, and split
into diacetyl by diacetyl synthetase. However, so far there was no direct evidence of
diacetyl synthetase. The second was proposed by Deman. The active acetaldehyde
was condensed with other pyruvate to form α-acetolactic acid by its synthetase
(Monnet et al. 1994), and then α-acetolactic acid was oxidized to form diacetyl
(Cogan et al. 1981). When in hypoxia, the outcome was 3-hydroxy-2-butanone and
then reduced to 2,3-butanediol with no obvious smell. Both of 3-hydroxy-2-
butanone and diacetyl had fragrant smell. The process of oxidative decarboxylation
had been supported by the data of biochemistry, genetics, and nuclear magnetic
resonance (Marth and Steele 1998). Diacetyl could be reduced by reductase to acet-
10 S. Wang et al.
oin (Cogan et al. 1981; Seitz et al. 1963). The catalytic activity of the enzyme had
great difference between different species or strains and, to a certain extent, was
affected by pH. Alpha-acetolactic acid was converted to acetoin by its decarboxyl-
ase. O’Sullivan et al. (2001) had successfully separated and purified the α-acetolactic
acid decarboxylase in Leuconostoc, determined the amino acid sequence, and con-
firmed the existence of the oxidative decarboxylation.
1.2.3.1.2 Production of Diacetyl by Glucose Metabolic Pathways
The metabolic pathways of diacetyl by lactic acid bacteria were mainly citric acid
metabolic and glycolytic pathways. Many Lactobacillus, especially Lactococcus,
could use lactose and citric acid to produce pyruvate and synthesize diacetyl
(Fig. 1.2). During the production of diacetyl from lactose, the excessive pyruvate
was used to synthesize α-acetolactic acid by its synthetase, and then diacetyl was
generated by oxidative decarboxylation reaction under the acidic condition. Diacetyl
was very unstable and could be reduced by reductase to acetoin. In the metabolic
pathways of citric acid to diacetyl, pyruvate and α-acetolactic acid were the most
important intermediates. Citric acid was lysed into oxaloacetic acid by lyase and
synthesizes to α-acetolactic acid from oxaloacetic acid directly or produced to pyru-
vate by decarboxylic reaction. Moreover, pyruvate decarboxylation reaction gener-
ated active acetaldehyde, which could be used with pyruvate to synthesize
α-acetolactic acid by its synthetase. And then diacetyl was generated by oxidative
decarboxylation reaction. Alpha-acetolactic acid was converted to acetoin by its
decarboxylase. Because of the low content of citric acid in milk, in general, diacetyl
was generated by glycolytic pathway in the fermented milk (Aymes et al. 1999;
Hugenholtz 1993; Curic et al. 1999).
During the process of glucose metabolism in Lactococcus, pyruvate was dehy-
drogenized by lactic dehydrogenase, became lactic acid, and transformed to acetyl
CoA by pyruvate dehydrogenase and formate lyase. Furthermore, acetyl CoA
became ethanol and acetaldehyde. Pyruvate was synthesized to α-acetolactic acid
Fig. 1.2 Synthesis pathways of acetaldehyde and diacetyl in Lactobacillus

by its synthetase. And diacetyl was generated by nonenzymatic natural oxidative

decarboxylation reaction under the acidic condition. Diacetyl could be reduced by
reductase to acetoin, which had been supported by a large number of biochemical
and molecular biological data. In recent years, more and more studies confirmed
that the main pathway was the nonenzymatic natural oxidative decarboxylation
reaction which made α-acetolactic acid to become diacetyl in Lactococcus (Snoep
et al. 1992).
1.2.3.2 Metabolic Regulation of Diacetyl in Lactic Acid Bacteria
1.2.3.2.1 Regulation of Culture Conditions
Production and stability of diacetyl depended on the growing environment of lactic

acid bacteria, including nutrient composition, temperature, pH, and the presence of
oxygen. Due to the established dairy technology, many of the external factors could
not be controlled in the fermented dairy products processing. Even in lactic acid
bacteria that could ferment citric acid, the capacity of diacetyl was relatively low in
dairy products. Researcher focused on the regulation of culture conditions to
increase the content of diacetyl in cheese and sour milk. In the production of dairy
products, citric acid fermentation strains were often mixed with acid-produced
strains to improve oxygen concentration and production of diacetyl (Boumerdassi
et al. 1997). In addition, oxygen could oxidize the NADH and decreased the reduc-
tive ratio of diacetyl to acetoin.
1.2.3.2.2 Regulation of α-Acetolactic Acid
The metabolism of pyruvate and citrate in Lactococcus and Leuconostoc confirmed

that diacetyl was produced with α-acetolactic acid by nonenzymatic natural oxida-
tive decarboxylation reaction. Alpha-acetolactic acid was transformed to acetoin by
α-acetolactic acid decarboxylase in Lactococcus, which irreversibly decreased
α-acetolactic acid. And the metabolism regulation of α-acetolactic acid was a pos-
sible way to improve the content of diacetyl. Based on the understanding of diacetyl
generation pathways, several metabolic engineering strategies have been designed
to increase the content of diacetyl in lactic acid bacteria. Because of the very low
content of citric acid in milk, researches all focused on the transformation of lactose
to diacetyl. Researchers had tried several methods as follows: devitalized the gene
of lactate dehydrogenase (LDH) (Gasson et al. 1996); overexpressed the genes of
ilvBN or alsS of α-acetolactic acid in anabolism and catabolism; devitalized the
genes of pyruvate lyase (Pfl2) and α-acetolactic acid decarboxylase (aldB) (Monnet
et al. 1997; Yang and Wang 1996); and overexpressed the gene of NADH oxidase
(nox) (Benson et al. 1996; Felipe et al. 1998). These strategies also could be com-
bined to make lactose and glucose efficiently transform into acetone, especially in
the inactivation of lactate dehydrogenase (LDH).
12 S. Wang et al.
The inactivation of LDH led to excessive accumulation of pyruvate. In theory,

the mutant strain produced more diacetyl than the wild strain. Studies had con-
firmed that site-specific mutation of LDH in Lactococcus was feasible to change the
pathway of glycometabolism. And the pyruvate couldn’t be converted to lactate but
to a mixed acid fermentation pathway with formic acid and alcohol as the main
product. Under aerobic conditions, the accumulation of pyruvate led to a large
amount of acetoin and butanediol.
Increasing the copy number of α-acetolactic acid gene and NADH oxidase gene
improved the content of α-acetolactic acid and eventually enhanced the production
of diacetyl (Benson et al. 1996; Felipe et al. 1998). Marugg et al. (1994) cloned and
expressed the gene of α-acetolactic acid synthase (als) and analyzed the character-
istics in diacetyl subspecies of Lactococcus lactis (Marugg et al. 1994). When the
gene of α-acetolactic acid synthase was devitalized or the enzyme was deficient in
Lactococcus lactis, more than 80% of the pyruvate was converted under the appro-
priate conditions.
One of the most promising ways to increase the production of diacetyl was to
inactivate the aldB gene, which leads to the accumulation of α-acetolactic acid as
the precursor of diacetyl (Goupil et al. 1996). The inactivation of α-acetolactic acid
decarboxylase directly led to the conversion of α-acetolactic acid to acetoin, there-
fore, increasing the diacetyl (Monnet et al. 1997; Yang and Wang 1996). Due to the
redox balance, sugar was converted to pyruvic and then to lactic acid. Meanwhile,
citric acid was converted to α-acetolactic acid and then was decarboxylated into
acetone. In dairy fermentation, through the chemical decarboxylation of α-acetolactic
acid, these bacteria could produce a large amount of diacetyl directly. New screen-
ing methods and genetic engineering had made the use of these bacteria possible.
The aldB mutant strain of Lactococcus could grow in leucine medium without add-
ing other branched-chain amino acids to achieve separation. The theoretical basis
was that the activity of Ald was regulated by allosteric modification of leucine,
which could directly activate Ald, and that α-acetolactic acid was the precursor of
leucine and valine. Most of lactococci irreversibly lost the ability to synthesize
branched-chain amino acids (Godon et al. 1993), and the growth was restricted in
the screening medium (Godon et al. 1993).
1.2.3.2.3 Regulation of Mixed Acid
Under different fermentation conditions, researchers studied the production of

diacetyl of Lactobacillus or its precursor, α-acetolactic acid, and the results showed
that the production of diacetyl was related to the utilization of citric acid. Because
of the low content of citric acid in foods, diacetyl could be produced efficiently with
sugars such as lactose or fructose.
In the production of dairy products, Lactobacillus had the potential to produce
various terminal products in the sugar fermentation, such as lactic acid, acetic acid,
diacetyl, acetaldehyde, or extracellular polysaccharide, which was beneficial to the
flavor and structure formation of dairy products and had important economic value.
Generally, Lactobacillus was the homolactic fermentation, and metabolic changes

have been found under certain conditions. For example, when Lactobacillus used
glucose as the sole carbon source and energy, under restrictive carbon sources or
aerobic conditions, the terminal products included other mixed acids besides lactic
acid. Recent studies had indicated that the decrease of glycometabolism rate led to
the variation of homolactic fermentation to mixed acid fermentation. Compared
with control, when high glycolysis was applied during metabolism under aerobic
conditions, it had been observed that metabolism flowed to mixed acid fermenta-
tion. Direct oxidation of NADH, which was required for pyruvate reduction, led to
a decrease of flowing to lactic acid via LDH. As an indicator of cellular redox, the
ratio of NAD+ to NADH was directly affected by the activity of NADH oxidase,
which determines the flow direction of metabolism.
Lactobacillus usually conducted glycometabolism through homolactic fermen-
tation and heterolactic fermentation. The terminal products of glycometabolism
could be produced by a variety of ways, not only included lactic acid in homozy-
gotic strain, the terminal products of glycometabolism could be produced by a vari-
ety of ways, not only included lactic acid (Fig. 1.3). Usually these fermentation
products were formed by the accumulation of pyruvate and the ratio of NADH to
NAD+. When the concentration of intracellular pyruvate exceeded lactic acid that
was produced by lactate dehydrogenase, it should supply other pathways of pyru-
vate removal, providing NADH oxidation. These alternate pathways also provided
the cells with the means to produce extra ATP. What conditions and circumstances
did the pyruvate will be accumulated? When substrate fermentation is limited, the
lack of fructose-1,6-diphosphate, low activity of isomerases, lactic dehydrogenase,
pyruvate would be accumulated. With high oxygen content in the environment,
NADH, which acted on the reduction of pyruvate, could be directly oxidized, pre-
venting lactate dehydrogenase reaction.
The enzymes and pathways were identified, which were involved in making pyru-
vate to form lactic acid or other products in Lactococcus and other Lactobacillus
(Cocaign-Bousquet et al. 1996; Garrigues et al. 1997). Under anaerobic conditions,
when carbohydrates were limited, low growth rate led to mixed acid fermentation,
forming alcohol, acetic acid, and formate. Pyruvate formate lyase was activated to
break down pyruvate into formate and acetyl CoA, which could be converted into etha-
nol and/or acetic acid. ATP also could be formed from acetic acid by acetokinase.
Fig. 1.3 Mixed acid fermentation of pyruvate

14 S. Wang et al.
Under aerobic conditions, the inactivation of pyruvate formate lyase led to the conver-
sion of pyruvate into acetic acid and CO2 by pyruvate dehydrogenase. Finally, excess
pyruvate was able to produce α-acetolactic acid by its synthase, and the reaction had
other important implications, because α-acetolactic acid was the precursor of diacetyl.
These alternative pathways of pyruvate metabolism were greatly influenced by
environmental conditions, and mutant strains that could not produce lactic dehydro-
genase must use excess pyruvate to produce other terminal products. Under certain
conditions, excess pyruvate might be converted to other products in cells, especially
flavor substance, such as diacetyl. Diacetyl was usually produced from citric acid,
but if the right conditions were established or the cells were generally modified,
cells that did not ferment citric acid could use lactose to form diacetyl. For example,
the overexpression of NADH oxidase in Lactococcus resulted in the reduction of
lactic acid from pyruvate while forming the precursor α-acetolactic acid of diacetyl
(Felipe et al. 1998). Studies had also found that glycometabolism of Lactobacillus
could be manipulated by changes in the concentration of pyruvate, an intermediate
product of metabolic engineering.
1.2.4 trategy for Increasing Production of Diacetyl

S
in Lactobacillus
1.2.4.1 ptimized the Culture Conditions to Increase the Yield
O
of Diacetyl
The yield and stability of diacetyl were affected by many external factors in the
fermentation of Lactococcus, such as the type and proportion of carbon and nitro-
gen sources in the culture medium, growth temperature, pH, oxygen, and so on.
Studies have found that under aerobic conditions, the yield of diacetyl was much
higher than that under anaerobic conditions (Curic et al. 1999). Under aerobic con-
ditions, the supplementation of citric acid in the medium could increase the yield of
diacetyl. The reason might be that under aerobic conditions, oxygen acted as an
electron receptor to promote the production of diacetyl during the process of the
formation through decarboxylation of α-acetolactic acid. And the supplementation
of citric acid led to further accumulation of pyruvate, resulting in the increased pro-
duction of diacetyl (Levata-Jovanovic and Sandine 1996). Therefore, in the fermen-
tation industry, in order to prevent the reduction reaction of diacetyl to form acetoin,
making fermentation dairy products lose their required flavor, the method of adding
citric acid or sodium citrate was often used to strengthen the milk to guarantee the
synthesis of diacetyl with constant and high yield. When it had achieved the desired
acidity and flavor, the product would be immediately cooled to inhibit the activity
of diacetyl reductase and decrease its damage to the diacetyl. In addition, it should
prevent the contamination of psychrophilic bacteria with high diacetyl reductase
activity. Optimization of culture conditions could improve the diacetyl production
capacity of Lactococcus to some extent but at the same time might affect the
proportion of other nutrients in the fermentation products and might produce some
harmful substances (Yang and Wang 1996).
1.2.4.2 pplication of Genetic Engineering and Metabolic Engineering

A
to Increase the Yield of Diacetyl
The yield of diacetyl in yogurt fermentation was closely related to the activity of
several key enzymes, including the citrate lyase, oxaloacetic decarboxylase, α-acetyl
synthase, NADH oxidase, α-acetolactate decarboxylase, diacetyl reductase, and so
on. These enzymes could be inactivated or overexpressed by using molecular biol-
ogy technology, which could increase the accumulation of the intermediate product
pyruvate, α-acetolactic acid, change the metabolic pathway of diacetyl, and increase
the yield of diacetyl. In order to change the metabolic pathway of α-acetolactic acid
to acetoin, some domestic and foreign scholars had focused on the screening and
construction of some strains with α-acetolactic acid decarboxylase deficiency.
Aymes et al. (1999) used random mutagenesis to screen the subspecies of
Lactococcus lactis butanedione with α-acetolactic acid decarboxylase deficiency,
and under anaerobic conditions, the yield of diacetyl was much higher than that
of the wild strain. In order to increase the yield of the diacetyl in streptococcus
thermophiles, Monnet and Corrieu (2007) used the method of directional mutation
to select the Streptococcus thermophilus with α-acetolactic acid decarboxylase defi-
ciency in the medium containing ketobutyric acid, leucine, isoleucine. Under the
microaerobic conditions, the yield of diacetyl in mutant strain was nearly 3 times
higher than that of the wild strain. On the basis of screening and construction of the
strain with α-acetolactic acid decarboxylase deficiency, some researchers overex-
pressed the key genes which regulated the production of diacetyl, and further
attempts were made to improve the yield of diacetyl by means of joint regulation.
Hugenholtz et al. (1993) found that the overexpression of nox gene regulating
NADH oxidase under aerobic conditions significantly increased the content of
diacetyl in the Lactococcus lactis with α-acetolactic acid decarboxylase deficiency.
At present, the gene regulation and metabolism of diacetyl in most studies focused
on the regulation of some key genes such as glycolysis, citric acid metabolic path-
way. With detailed studies, more new products should be produced that meet the
demands of consumers.
1.2.5 Application of Diacetyl in Foods
Diacetyl inhibited many microorganisms, including spoilage and pathogenic bacte-

ria. Low concentrations (several milligrams per liter) of diacetyl inhibited the
growth of E. coli, and the antibacterial activity of diacetyl increased as pH decreased.
Some scholars had confirmed the synergistic effect of diacetyl antibacterial activity
at low pH (Jay et al. 1992). Diacetyl inhibited the growth of gram-negative bacteria
16 S. Wang et al.
by interfering with the use of arginine by reacting with the arginine-binding protein
of gram-negative bacteria.
Diacetyl was considered to be one of the most important ingredients in the flavor
of dairy products, as well as an important flavor in butter, cream, cheese, and many
nondairy products that require milk flavor (Hugenholtz et al. 2000). Most
Lactobacillus such as Lactococcus lactis, Streptococcus lactis, and Streptococcus
thermophilus could produce diacetyl (Aymes et al. 1999). However, diacetyl was
the main factor affecting the maturation period of beer flavor in beer fermentation.
When the content exceeded the threshold (0.15 × 10–6), it would produce an
unpleasant sour beer taste (Bartowsky and Henschke 2004).
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Chapter 2
Lactic Acid Bacteria and Conjugated Fatty
Acids
Wei Chen, Bo Yang, and Jianxin Zhao
2.1 Introduction
Conjugated fatty acid (CFA) refers to a group of positional and geometric isomers
of polyunsaturated fatty acid possessing conjugated double bonds. Conjugated dou-
ble bonds, conjugated triple bonds, and conjugated quadruple bonds are the typical
conjugated fatty acid forms, in which conjugated octadecadienoic acid and conju-
gated octadecatrienoic acid are most common isomers, such as conjugated linoleic
acid (CLA), conjugated linolenic acid (CLNA), and conjugated steariconic acid
CSA (Yang et al. 2015).
2.1.1 Conjugated Linoleic Acid
Conjugated linoleic acid (CLA) is a generic term of octadecadienoic acid with con-
jugated double bonds, referring to a group positional and geometric isomer of lin-
oleic acid (LA), in which each conjugated double bond exists in two types, cis (c)
and trans (t). In theory, according to the position of double bonds, 54 isomers of
CLA could be synthesized; however, until now, only 28 isomers have been identi-
fied, including conjugated double bond on C7,C9, C8,C10, C9, C11, C10,C12, C11, and
C13. c9, t11-CLA (rumenic acid) was the most abundant CLA isomer, followed by
t10,c12-CLA (Andrade et al. 2012).
CLA has attracted much attention due to its physiological effects, such as anti-
inflammation, anticancer, reduction of atherosclerosis, anti-obesity, amelioration of
W. Chen (*) · B. Yang · J. Zhao

Jiangnan University, Wuxi, China
e-mail: chenwei66@jiangnan.edu.cn; jxzhao@jiangnan.edu.cn
© Springer Nature Singapore Pte Ltd. and Science Press 2019 21

W. Chen (ed.), Lactic Acid Bacteria,
https://doi.org/10.1007/978-981-13-7283-4_2
22 W. Chen et al.
Fig. 2.1 Structure of linoleic acid and major CLA isomers
diabetes, promotion of bone growth, and immune regulation. As reported, the bio-
logical function was isomer-dependent, in which c9,t11-CLA and t10,c12-CLA
were recognized as the CLA isomers with best physiological effects (Fig. 2.1). The
major physiological functions of c9,t11-CLA were anti-cancer, anti-inflammation,
and immune regulation, whereas t10,c12-CLA has significant benefits on anti-
obesity and regulation of lipid metabolism. Additionally, t9,t11-CLA was reported
with anti-inflammation function (Yang et al. 2015).
CLA naturally occurs in ruminant milk and issues; therefore, ruminant dairy and
meat products are the main source of CLA in the daily diet, in which c9, t11-CLA
comprised of 80–90% fatty acid of the total dairy lipids and t10,c12-CLA comprised
of only 1% of total dairy lipids. Moreover, other CLA isomers, such as t7,c9-CLA,
c8,t10-CLA, t10,c12-CLA, and t11,c13-CLA, could be detected in the milk (Jensen
2002). In ruminant animals, two major sources of CLA were reported: (1) CLA was
mainly produced as one of the intermediates by some ruminant bacteria in the pro-
cess of catalyzing LA into stearic acid (C18:0), and (2) numerous researches have
reported that c9,t11-CLA could be generated by Δ9-dehydrogenase in the mammary
gland with vaccenic acid (t11-C18:1) as substrate. Many studies on the CLA-
producing mechanism in ruminant bacteria have been carried out (Kepler et al. 1966,
1971; Kepler and Tove 1967; Polan et al. 1964; Rosenfeld and Tove 1971) that LA
could be quickly transformed into CLA by linoleic acid isomerase in ruminant bac-
teria and then transferred into vaccenic acid at a slower rate. After vaccenic acid was
accumulated to a certain level, it would be further transformed to stearic acid. Other
studies have demonstrated that some vaccenic acid in ruminant animals could be
absorbed and then transported to other tissues. Vaccenic acid in the mammary bland
could be further transferred into CLA through catalyzing by Δ9-dehydrogenase
(Bauman et al. 2001). It has been identified that CLA generated from this process
could comprise of 60–70% of total CLA in the milk (Corl et al. 2001).
2 Lactic Acid Bacteria and Conjugated Fatty Acids 23
2.1.2 Conjugated Linolenic Acid
Conjugated linolenic acid (CLNA) was one of the derivates from linolenic acid
(LNA, C18:3) with conjugated double bonds, comprising of different isomers
(Fig. 2.2). CLNA was firstly discovered in the nineteenth century; however, it was
not attracted much attention due to rare awareness of its physiological effects. Till
1987, Nuteren and Christ-Hazelhof firstly identified the biological activity of CLNA
when they studied the inhibitory effect of fatty acids derived from plant seeds on the
synthesis of prostaglandin E2 (PGE2) (Nugteren and Christ 1987). Later, anti-
cancer and anti-obesity activities of naturally CLNA from bifidobacteria were
reported by other researchers (Coakley et al. 2009; Hennessy et al. 2012; Destaillats
et al. 2005.) The bifidobacterial CLNA isomers analyzed included c9,t11,c15-
CLNA, t9,t11,c15-CLNA, c9,t11,c13-CLNA, c9,t11,t13-CLNA, c6,c9,t11-CLNA,
and c6,t9,t11-CLNA.
CLNA was widely distributed in nature, such as milk and ruminant meet. In
addition, CLNA occurs in some plant seeds, for instance, pomegranate seeds, tung
oil seeds, momordica charantia seeds, calendula seeds, etc. The CLNA isomers
derived from plant seeds consist of many kinds of isomers, and thus proper
Fig. 2.2 Structure of α-linolenic acid, γ- linolenic acid, and conjugated linolenic acids
24 W. Chen et al.
Fig. 2.3 Structure of conjugated stearidonic acids
separation methods would be key factors to obtain the pure isomers (Smith Jr.
1971). Recently, supercritical CO2 fluid extraction and low-temperature-crystalliza-
tion methods have been applied to separate CLNA. However, recent separation
methods could not address the commercial requirements due to the limited amount
of food grade plants.
2.1.3 Other Conjugated Fatty Acid
Despite CLA and CLNA, another conjugated fatty acid is conjugated stearidonic
acid (CSA). CSA has been identified to possess many physiological activities, such
as anti-tumor, antiatherosclerosis, and hypoglycemic activity. Besides, CSA could
be applied in lipid peroxidation to evaluate the antioxidant agents. The identified
isomers of CSA include c6,c9,t11,c15-CSA, c6,t9,t11,c15-CSA, c9,t11,t13,c15-
CSA, and t9,t11,t13,c15-CSA (Fig. 2.3) (Hennessy et al. 2012).
2.2 actic Acid Bacteria with Conjugated Fatty Acid

L
Production Ability
Microbial CFA producers have been studied for decades, which started in 1960s.
Kepler et al. (1966, 1970, 1971), Kepler and Tove (1967) originally found that
Butyrivibrio fibrisolvens, one of the ruminant bacteria, could convert LA to
CLA. Then a variety of microbes showed the property of CLA production, espe-
cially that lactic acid bacteria could generate c9,t11-CLA and t9,t11-CLA. With the
increase of research of CLA production in lactic acid bacteria, CLNA, CSA, and
2 Lactic Acid Bacteria and Conjugated Fatty Acids 25
other CFA were found in lactic acid bacteria metabolites. Lactic acid bacteria with
CFA production ability include Lactobacillus (L. plantarum, L. acidophilus, L.
casei, L. reuteri, L. fermentum, L. bulgaricus, L. rhamnosus), Bifidobacterium (B.
breve, B. longum, B. animalis subsp. lactis), Lactococcus lactis, Streptococcus ther-
mophiles, etc.
2.2.1 Conjugated Fatty Acid Production in Lactobacilli
Lactobacillus was widely reported with CFA production ability, especially CLA
production, which consisted of almost each species of lactobacilli (Table 2.1).
2.2.1.1 Lactobacillus plantarum
L. plantarum was the most widely studied strain among lactobacilli with CLA-
production ability. In 2002, Kishino et al. screened many lactic acid bacteria strains
with CLA-production ability, including Lactobacillus, Enterococcus, Pediococcus,
and Propionibacterium (Kishino et al. 2002), in which L. plantarum AKU1009a
was the strain with the best CLA-generation ability. Further study demonstrated that
L. plantarum AKU1009a could even transform ricinoleic acid into CLA directly.
Interestingly, the washed cells of this strain could catalyze α-linolenic acid into
c9,t11,c15-CLNA and t9,t11,c15-CLNA with a 40% of total conversion rate.
Comparatively, it has a better ability of CLNA from γ-linolenic acid with a conver-
sion rate up to 68%. CSA could be produced from stearidonic acid into c6,c9,t11,c15-
CSA and c6,t9,t11,c15-CSA by the strain (Kishino et al. 2010). The concentration
of ricinoleic acid utilized by the washed cells of L. plantarum JCM1551 was up to
2400 mg/L with c9,t11-CLA and t9,t11-CLA as the main isomers (Andrade et al.
2012). L. plantarum NCUL005 has also been reported to produce CLA with final
concentration of 623 mg/L growing in MRS medium in the presence of free LA
(Andrade et al. 2012). Furthermore, growing and washed cells of L. plantarum
ZS2058 could both transform free LA into CLA with conversion rate as 54.3% and
46.75%, respectively (Yang et al. 2017).
Except growing cells in the MRS medium or washed cells in the proper reaction
solution, the strains added into the fermentis medium including sunflower oils or
soymilk could also be used as the CLA producers (Li et al. 2012). VSL3# was the
most widely used probiotics including eight strains, and studies have reported that
all the eight strains could produce CLA, in which the conversion rate of L. planta-
rum strain was about 60% with c9, t11-CLA and t9,t11-CLA as the main isomers
(Ewaschuk et al. 2006).
Furthermore, the CLA production mechanism by lactobacilli was also identified.
Shimizu et al. firstly found that LA was firstly transformed into 10-hydroxy-cis-12
octadecenic acid and 10-hydroxy-trans-12-octadecenic acid, and then these two
intermediates were both transferred into CLA (Ogawa et al. 2001). Further analysis
Another random document with
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. . . . .
The following thoughtful description of the action appeared in the German
wireless communiqué next morning:
“Our light forces in an enterprise off the English coast put to flight a vastly
superior strength of armed merchant cruisers escorted by destroyers. English fleet
on coming to the rescue was compelled to withdraw, and our forces returned to
harbour without further molestation.”
Every man to his own trade.
III. The Left Flank

The north-east wind carried the steady grumble of gunfire across the sand-dunes
and far out to sea.
The foremost gun’s crew of a British destroyer stood huddled in the lee of the
gun-shield with their duffle hoods pulled down over their foreheads. The sea was
calm, and the stars overhead shone with frosty brilliance. A figure groped its way
forward with a bowl of cocoa, and joined the group round the breech of the gun.
They drank in turn, grunting as the warmth penetrated into their interiors.
The distant gunfire swelled momentarily. Above the horizon far ahead
intermittent gleams marked the activity of searchlight and star-shell.
“Them’s our guns,” said one of the cocoa-drinkers. He wiped his mouth on the
sleeve of his coat, and stared ahead. It never seemed to occur to any of them that
they might equally well be German guns.
“That’s right,” confirmed the sight-setter. “There’s guns going like that for
’undreds an’ ’undreds of miles. Right away up from the sea. Me brother’s there—
somewhere....” For a moment they ruminated over a mental picture of the sight-
setter’s brother, a mud-plastered stoical atom, somewhere along those hundreds of
miles of wire and bayonets that hedged civilisation and posterity from the
Unnamable. “Switzerland to the sea,” said the speaker. He jerked the breech-lever
absent-mindedly towards him, and closed it again with a little click.
“An’ then we takes on,” said a loading number. “Us an’ these ’ere.” He tapped
the smooth side of a lyddite shell lying in the rack beside him.
“An’ this ’ere,” said the man who had brought the cocoa. He thrust forward the
cumbersome hilt of a cutlass at his hip. The starlight gleamed dully on the steel
guard.
“You won’t use that to-night, my son,” said the gunlayer. “We ain’t goin’ to
’ave no Broke an’ Swift song an’ dance to-night.” He stared out into the clear
darkness. “We couldn’t never git near enough.” Nevertheless, he put out his hand
in the gloom and reassured himself of the safety of a formidable bar of iron well
within reach. Once in the annals of this war had a British destroyer come to grips at
close quarters with the enemy; thereafter her crew walked the earth as men apart,
and the darlings of the high gods.
The night grew suddenly darker. It was the mysterious hour that precedes the
dawn, when warring men and sleeping animals stir and bethink them of the
morrow. The destroyer slackened speed and turned, the wide circle of her wake
shimmering against the darkness of the water. As they turned, other dark shapes
were visible abeam, moving at measured distance from each other without a light
showing or a sound but the faint swish of the water past their sides. The flotilla had
reached the limit of its beat, and swung round to resume the unending patrol.
Once from the starry sky came the drone of a seaplane moving up from its base
that lay to the southward. Another followed, another and another, skirting the coast
and flying well out to sea to avoid the searchlights and anti-aircraft guns of the
shore batteries. They passed invisible, and the drone of their engines died away.
“Our spottin’ machines,” said the sight-setter. “They’re going up to spot for the
monitors at daylight.” He jerked his head astern to the north, and yawned. “I
reckon I’d sooner ’ave this job than screenin’ monitors wot’s bombardin’ Ostend. I
don’t fancy them 15-inch German shell droppin’ round out o’ nuffink, an’ no
chance of ’ittin’ back.”
“They knocks seven-bells outer Ostend, them monitors,” said another. “We ain’t
knockin’ ’ell out o’ nobody, steamin’ up an’ down like one of them women slops in
the ’Orseferry Road.” The speaker blinked towards the east where the stars were
paling.
“We’re all doin’ our bit,” said the gunlayer, “an’ one o’ these nights....” He
shook his head darkly. The dawn crept into the sky: the faces beneath the duffle
hoods grew discernible to each other, unshaven, pink-lidded, pinched with cold.
Objects, shining with frozen dew, took form out of the black void. The outline of
the bridge above them, and the mast behind, stood out against the sky; the head and
shoulders of the captain, with his glasses to his eyes, appeared above the bridge
screen, where he had been all night, watchful and invisible. The smoke trailing
astern blotted out the rest of the flotilla following in each other’s wake. Aft along
the deck, guns’ and torpedo-tubes’ crews began to move and stamp their feet for
warmth.
Away to starboard a circular object nearly awash loomed up and dropped astern.
Another appeared a few minutes later, and was succeeded by a third. Mile after
mile these dark shapes slid past, stretching away to the horizon. They were the
buoys of the Channel barrage, supporting the mined nets which are but a
continuation of four hundred miles of barbed wire.
The day dawned silvery grey and disclosed a diffused activity upon the face of
the waters. Two great hospital ships, screened by destroyers as a sinister reminder
to the beholder of Germany’s forfeited honour, slid away swiftly towards the
French coast. A ragged line of coastwise traffic, barges under sail, lighters in tow
of tugs, and deepladen freighters hugging the swept channel along the coast,
appeared as if by magic out of “the bowl of night”; from the direction of the chalk-
cliffs came a division of drifters in line ahead. They passed close to the destroyers,
and the figure on the leading destroyer’s bridge bawled through a megaphone.
They were curt incoherencies to a landsman—vague references to a number and
some compass bearings. A big man on board the drifter flagship waved his arm to
indicate he understood the message; which was to the effect that one of the barrage
buoys appeared to have dragged a little, and the net looked as if it was worth
examining.
The drifters spread out along the line of buoys and commenced their daily task
of overhauling the steel jackstays, testing the circuits of the mines, repairing
damage caused by the ebb and flow of the tide and winter gales.
Half an hour later the destroyers encountered their reliefs, transferred the
mantle of responsibility for the left flank with a flutter of bunting and a pair of
hand-flags, and returned to their anchorage, where they were greeted by a
peremptory order from the signal station to complete with oil fuel and report when
ready for sea again. A coastal airship had reported an enemy submarine in the
closely guarded waters of the Channel, and along sixty miles of watchful coast the
hunt was up.
“My brother Alf,” said the sight-setter disgustedly, as he kicked off his seaboots
and prepared for an hour’s sleep, “ ’e may be famil’r wif tools wot I don’t know
nothin’ about. But there’s one thing about ’em—when ’e lays ’em down, ’e bloody-
well lays ’em down.”
IV. The Hunt

The Blimp rose from her moorings, soaring seaward, and straightway the roar of
her propeller cut off each of the occupants of the car into a separate world of his
own silence. The aerodrome with its orderly row of hangars dropped away from
under them with incredible swiftness. Fields became patchwork, buildings fell into
squares and lozenges without identity. Figures which a minute or two before had
been noisy, muscular, perspiring fellow-men working on the ropes, were dots
without motion or meaning, and faded to nothingness.
A flock of seagulls rose from the face of the cliff, whirled beneath them like
autumn leaves, and dropped astern. The parallel lines of white that were breakers
chasing each other to ruin on a rock-bound coast merged into the level floor of the
Atlantic, and presently there was nothing but sea and blue sky with the rushing
wind between, and this glittering triumph of man’s handiwork held suspended like
a bauble midway.
The pilot turned in his seat and grinned over his shoulder at the observer. The
grin was the only visible portion of his face: the rest was hidden by flying-helmet
and goggles and worsted muffler. The grin said: “It’s a fine morning and the old
bus is running like a witch. What’s the odds on sighting a Fritz?”
The observer laughed and shouted an inaudible reply against the roar of the
wind. He pulled a slip of chewing gum out of his pocket, bit off a piece, and passed
the rest to the pilot. Then he adjusted the focus of the high-power glasses and
began methodically quartering out the immense circular expanse of sea beneath
them.
Half an hour had passed when the wireless operator in the rear leaned forward
and tapped him on the shoulder. His listeners were to his ears, and he was
scribbling something on a slate. “S.O.S.—S.O.S.”—a bearing from a distant
headland—“fourteen miles—S.O.S.—S.O.S.—come quickly—I am being shelled
—S.O.S.—Subma——” The operator paused with his pencil above the slate,
waited a moment, and handed the slate forward.
The message, soundless, appealing, that had reached them out of the blue
immensity had ceased abruptly. The pilot glanced from the compass to a small
square of chart clamped before him, and slowly turned the wheel. Then he looked
back over his shoulder and grinned again.
A quarter of an hour later the pilot extended a gauntletted hand and pointed to
the rim of the horizon. A faint smudge of smoke darkened into a trailing cloud, and
presently they saw ahead of it the forepart of a ship, driving through the water at a
speed which clove a white, irregular furrow across the surface of the sea. She was
swerving from side to side like a hunted buck, and as the dirigible dipped her nose
and the hum of the wind redoubled to a roar in the ears of the crew, they saw away
to the west a tiny cigar-shaped object. At intervals a spurt of flame shot from it, and
a little pale mushroom-shaped cloud appeared above the steamer as the shrapnel
burst.
The Blimp swooped at eighty miles an hour upon that cigar-shaped object. The
observer braced his feet and grasped the bomb release lever, his jaws still moving
about the piece of chewing-gum. The sea, flecked with little waves, rushed up to
greet them. They had a glimpse of the submarine’s crew tumbling pell-mell for the
conning-tower hatchway, of the wicked gun abandoned forward still trained on the
fleeing merchantman. The next instant the quarry had shot beneath them. A sharp
concussion of the air beat upon the fragile car and body of the airship as her nose
was flung up and round. The dirigible’s bomb had burst right forward on the
pointed bows, and the submarine was diving in a confused circle of broken water
and spray.
The Blimp turned to drop another bomb ahead of the rapidly vanishing wake,
and then marked the spot with a calcium flare, while the wireless operator jiggled a
far-flung “Tally ho!” on the sending-key of his apparatus.
The tramp disappeared below the horizon, and they caught disjointed scraps of
her breathless tale while they circled in wide spirals above the watery arena.
Three motor launches were the first upon the scene, each with a slim gun in the
bows, and carrying, like hornets, a sting in their tails. They were old hands at the
game, and they spread out on the hunt with business-like deliberation under the
directions of the Blimp’s Morse lamp. The captain of the inshore boat (he had been
a stockbroker in an existence several æons gone by) traced a tar-stained finger
across the chart, and glanced again at the compass. “Nets—nets—nets,” he
mumbled. “The swine probably knows about those to the northwest ... He daren’t
go blind much longer. Ha!”
“Feather three points on your port bow,” winked the Blimp. Over went the
motor launch’s helm, and the seaward boat suddenly darted ahead in a white cloud
of spray. Bang! a puff of smoke drifted away from the wet muzzle of her gun; half
a mile ahead a ricochet flung up a column of foam as the shell went sobbing and
whimpering into the blue distance.
“Periscope dipped,” waved a pair of hand-flags from the boat that had fired.
And a moment later, “Keep out of my wake! Am going to release a charge.”
For an hour that relentless blindfold hunt went forward, punctuated by
exploding bombs and depth charges, and the crack of the launches’ guns as the
periscope of the submarine rose for an instant’s glimpse of his assailants and
vanished again. Twice the enemy essayed a torpedo counter-attack, and each time
the trail passed wide. Then, crippled and desperate, he doubled on his tracks, and
for a while succeeded in shaking off the pursuit. Nets, as he knew, lay ahead, and
nets were death; safety lay to the southward could he but keep submerged; but the
water, spurting through the buckled plating and rivets started by the bursting depth
charges, had mingled with the acid in the batteries and generated poison gas, which
drove him to the surface. Here he turned, a couple of miles astern of his pursuers,
and manned both guns, a hunted vermin at bay. As his foremost gun opened fire, a
heavy shell burst a few yards abeam of the submarine, and the captain of the
nearest motor launch raised his glasses. It was not a shell fired from a motor
launch.
“The destroyers,” he said. “Now why couldn’t they have kept away till we’d
made a job of it?” On the horizon the masts and funnels of a flotilla of destroyers
appeared in line abreast, approaching at full speed, firing as they came. The next
instant a shell from the submarine burst on the tiny forecastle of the launch,
shattering the gun, gun’s crew, and wheelhouse. The coxswain dropped over the
wrecked wheel and slowly slid to the deck like a marionette suddenly deprived of
animation. The lieutenant R.N.V.R. who had once been a stockbroker stood upright
for an instant with his hands to his throat as if trying to stem the red torrent
spurting through his fingers, and then pitched brokenly beside the coxswain.
The captain of the submarine counted the approaching destroyers, opened the
seacock to speed the flooding of his doomed craft, gave a swift glance overhead at
the Blimp swooping towards them for the coup-de-grâce, and ordered Cease Fire.
Then he waved his hands in token of surrender.
V. Overdue
The thin light of a sickle moon tipped the crest of each swift-running sea with
silver. The rest was a purple blackness, through which the north wind slashed like a
knife, and the sound of surf on a distant shoal was carried moaning. At intervals a
bank of racing clouds trailed across the face of the moon, and then all was inky
dark for a while.
It was during one of these periods of obliteration of all things visible that a
slender, perpendicular object rose above the surface of the sea. Gradually the dim
light waxed again: a wave, cloven in its path, passed hissing on either side in a trail
of spray, and the object slowly projected until it topped the highest wave. Presently
about its base a convulsive disturbance in the water was followed by the
appearance of a conical shape, a solid blackness against the streaked glimmering
obscurity of the water breaking all about its sides and streaming in cascades from
the flat railed-in top. A hatchway opened, and two figures crawled out, clinging to
the rail of their swaying foothold while the full force of the wind clawed and
battered at their forms. They maintained a terse conversation by dint of shouting in
turn with their lips to each other’s ears, while the conning-tower of the submarine
on which they stood moved forward in the teeth of the elements.
For half an hour they went plunging and lurching onwards, clinging with
numbed hands to the rail as a green sea swept about their legs, wiping the half-
frozen spray from their eyes to search the darkness ahead with night-glasses. Then
one pointed away on the bow.
“How’s that?” he bawled. A point on the bow something dark tumbled amid the
waves and flying spindrift. The other stared a moment, shouted an order to the
invisible helmsman through a voice-pipe, and the wind that had hitherto been in
their streaming eyes smote and buffeted them on the left cheek. A scurry of sleet
whirled momentarily about them, blotting out the half-glimpsed buoy; the taller of
the two figures put out an arm and smote his companion on the back. They had
made that buoy at dawn the previous day, and then, according to the custom of
British submarines in enemy waters, submerged till nightfall. Now, despite the set
of the tides and currents and the darkness, they had found it again, and with it their
bearings for the desperate journey that lay ahead.
For two hours they groped their way onwards through what would have been
unfathomable mysteries to a landsman. Compass, chart, and leadline played their
part: but not even these, coupled with the stoutest heart that ever beat, avail against
unknown minefield and watchful patrols. Thrice the two alert, oilskin-clothed
figures dived through the hatchway into the interior of the submarine, and the
platform on which they had been standing vanished eerily beneath the surface as a
string of long, dark shapes went by with a throb of unseen propellers. Once when
thus submerged an unknown object grated past the thin shell with a harsh metallic
jar, and passed astern in silence. Then it was that the captain of the submarine
removed his cap, passing his sleeve quickly across his damp forehead, and the
gesture was doubtless accepted where all prayers of gratitude find their way.
The first gleam of dawn, however, found no submarine on the surface. It
showed a business-like flotilla of destroyers on their beat, and a long line of net
drifters at anchor in the far distance amid sandbanks. An armed trawler with rust-
streaked sides and a gun forward
was making her way through the
cold, grey seas in the direction
of the drifters; a hoist of gay-
coloured signal flags flew from
her stump of a mast, and at the
peak a tattered German ensign.
The crew were clustered for
warmth in the lee of the engine-
room casing, their collars turned
up above their ears, and their
hands deep in their pockets.
They were staring ahead intently
at the line of nets guarding the
entrance to the harbour they
were about to enter. None
noticed a black speck that
peeped intermittently out of their
tumbling wake thirty yards
astern, and followed them up the
channel. Three or four fathoms
beneath that questioning speck,
in an electric-lit glittering steel
cylinder, a young man stood
peering into the lens of a high-
power periscope, both hands
resting on a lever. He spoke in a
dull monotone, with long
intervals of silence; and
throughout the length of that
cylinder, beside valve and dial
and lever, a score of pairs of
eyes watched him steadfastly.
“She’s given her funnel a
coat of paint since last month ...
port ten—steady! steady!...
There’s the gate vessel
moving.... The skipper is waving
to hurry him up.... Wants his
breakfast, I suppose.... That
must be the big crane in the dockyard.... There are flags hung about everywhere....
Starboard a touch.... It’s getting devilish light.... There’s something that looks like a
battle-cruiser alongside....”
There was a long silence, then the figure manipulating the periscope suddenly
stood upright.
“We’re through,” he said quietly. “And that’s their new battle-cruiser.”
. . . . .
In the smoking-room of a British submarine depot a group of officers sat round
the fire. Now and again one or other made a trivial observation from behind his
newspaper; occasionally one would glance swiftly at the clock and back to his
paper as if half afraid the glance would be intercepted. The hands of the clock crept
slowly round to noon; the clock gave a little preliminary whirr and then struck the
hour.
“Eight bells,” said the youngest of the group in a tone of detachment, as if the
hour had no special significance. A grave-faced lieutenant-commander seated
nearest the door rose slowly to his feet and buttoned up his monkey jacket.
“You goin’, Bill?” asked his neighbour in a low voice.
The upright figure nodded. “He’d have done as much for me,” he replied, and
walked quickly out of the room.
No one spoke for some minutes. Then the youngest member lowered the
magazine he was holding in front of him.
“Do they cry?” he asked.
“No,” said two voices simultaneously. “ ’Least,” added one, “not at the time.”
The silence settled down again like dust that had been disturbed; then the first
speaker leaned forward and tapped the ashes out of his pipe.
“Well,” he observed, “they didn’t get him cheap, at all events. I’m open to a bet
that he sent a Boche or two ahead of him to pipe the side.”
The group nodded a grim assent.
“Yes,” said one who had not hitherto spoken. “I reckon you’re right. But we
shan’t hear till the war’s over. They know how to keep their own secrets.” He
puffed at his pipe reflectively.
“Anyhow, thank God I’m a bachelor,” he concluded. He lifted a fox-terrier’s
head between his hands and shook it gently to and fro. “No one need go and tell
our wives if we don’t come back—eh, little Blinks?” The dog yawned, gave the
hands that held him a perfunctory lick, and resumed his interrupted nap sprawling
across his master’s knees.
. . . . .
Among the letters intercepted shortly afterwards on their way to a South
American State from Germany was one that contained the following significant
passage:
“ ... Yesterday all Kiel was beflagged: we were to have had a half-holiday on
the occasion of the trials of the great new battle cruiser——. Owing to an
unforeseen incident, however, the trials were not completed. Our half-holiday has
been postponed indefinitely....”
VI. “Tuppence Apiece”

The herring were in the bay, and the fleet of sailing smacks went trailing out on
the light wind with their eager crews of old men and boys straining at the halliards
to catch the last capful of wind. After them came the armed guard-boat of the little
peaceful fleet, a stout trawler with a gun in her bows, fussing in the wake of her
charges.
The skipper of the guard-boat was at the wheel, a tall, gaunt old man with a
fringe of grey whisker round his jaws and a mouth as tight as a scar. He it was who
located the herring and placed the fleet across their path, and all that day the
smacks lay to their nets till the porpoises turned inshore and drove the silvery host
eastward. After them went the smacks, with holds half-full, lured on by the
promise of two quarters’ rent as good as paid. Finally, the old Trawler Reserveman
checked the pursuit.
“Fish or no fish,” he cried. “Here ye
bide the night.” They had reached the limit of the safety zone in those waters, and
he rounded up his flock like a sagacious sheep-dog, counting the little craft
carefully ere he took up his position to seaward of them for the night. At the first
hint of dawn he weighed anchor and counted again: his grim old face darkened. He
turned to seaward where the sky was lightening fast, and searched the mist through
glasses. Three smacks were discernible some miles outside their allotted area. The
burly mate stood beside his father, and watched the delinquents hauling in their
nets with a speed that hinted at an uneasy conscience.
“They’m drifted in a bit of a tide rip, mebbe?” he ventured.
The old man growled an oath. “Tide rip? Nay! They’m just daft wi’ greed.
There’s no wit nor dacency in their sodden heads. An’ I’ll larn ’em both. By God
I’ll larn ’em to disobey my orders.” ... He watched the far-off craft hoisting sail,
with eyes grey and cold as flints beneath the bushy brows. “Aye,” he said
threateningly, “I’ll larn ye ...” and clumped forward to the wheel-house.
The sun had not yet risen, and the thin morning mists wreathed the face of the
waters. As the trawler gathered way a sudden flash of light blinked out of the mist
to the northward. The report of a gun was followed by the explosion of a shell fifty
yards on the near side of the most distant fishing-smack.
The trawler skipper measured the distance from the flash to the fishing fleet,
and thence to the truants bowling towards them on the morning breeze.
“Man the gun!” he roared. “Action Stations, lads!” He picked up a megaphone
and bellowed through it in the direction of his charges: “Cut your warps an’ get ter
hell outer this!” Then he wrenched the telegraph to full speed and put the wheel
over, heading his little craft towards the quarter from which the flash had come.
The gun’s crew closed up round the loaded gun, rolling up their sleeves and
spitting on their hands as is the custom of their breed before a fight.
“There’s a submarine yonder in the mist,” shouted the skipper. “Open fire
directly ye sight her and keep her busy while the smacks get away.” Astern of them
the small craft were cutting their nets away and hoisting sail. Three or four were
already making for safety to the westward before the early morning breeze that
hurried in catspaws over the sea.
Bang!
The trawler opened fire as the submarine appeared ahead like a long, hump-
backed shadow against the pearly grey of the horizon. The breech clanged open
and the acrid smoke floated aft as they reloaded.
“Rapid fire!” shouted the skipper. Shells were bursting all about the fleeing
smacks. “Give ’em hell, lads. Her’ve got two guns an’ us but the one....” He
glanced back over his shoulder at the little craft he was trying to save, and then
bent to the voice-pipe. “Every ounce o’ steam, Luther. Her’ll try to haul off an’
outrange my little small gun.”
Smoke poured from the gaily-painted funnel; the “little small gun” barked and
barked again, and one after the other the empty cylinders went clattering into the
scuppers. A shell struck the trawler somewhere in the region of the mizzen mast,
and sent the splinters flying. A minute later another exploded off the port bow,
flinging the water in sheets over the gun’s crew. The sight-setter slid into a sitting
position, his back against the pedestal of the gun-mounting, and his head lolling on
his shoulder. They had drawn the enemy’s fire at last, and every minute gave the
smacks a better chance. Shell after shell struck the little craft as she blundered
gallantly on. The stern was alight: the splintered foremast lay across a funnel
riddled like a pepper-pot. The trawler’s boy—a shock-headed child of fourteen
who had been passing up ammunition to the gun—leaned whimpering against the
engine-room casing, nursing a blood-sodden jacket wrapped about his forearm.
The mate was at the gun, round which three of the crew lay. One had raised
himself on his elbow and was coughing out his soul. The other two were on their
backs staring at the sky.
In the face of the trawler’s fire, the submarine turned and drew out of range,
firing as she went. One of the British shells had struck the low-lying hull in the
stern, and a thin cloud of grey smoke ascended from the rent. Figures were visible
running aft along the railed-in deck, gesticulating.
“Ye’ve hit her,” shouted the skipper from the wheel. “Give ’em hell, lads——”
A sudden burst of flame and smoke enveloped the wheel-house, and the skipper
went hurtling through the doorway and pitched with a thud on the deck.
The mate ran aft and knelt beside him. “Father,” he cried hoarsely.
The inert blue-clad figure raised himself on his hands, and his head swayed
between his massive shoulders.
“Father,” said the mate again, and shook him, as if trying to awaken someone
from sleep. “Be ye hurted terrible bad?”
The grim old seadog raised his head, and his son saw that he was blind.
“Pitch the codes overboard,” he said. “I’m blind an’ stone deaf, an’ my guts are
all abroad under me, but ye’ll fight the little gun while there’s a shell left
aboard....”
The mate stood up and looked aft along the splintered, bloody deck, beyond the
smoke and steam trailing to leeward.
“The gun’s wrecked,” he said slowly, as if speaking to himself. “The little
smacks are clear o’ danger.... The destroyers are comin’ up.... Ye have fought a
good fight, father.” The submarine had ceased fire, and as he spoke, she submerged
and vanished sullenly, like a wild beast baulked of its prey.
. . . . .
An old woman sat knitting beside the fire in the heart of a Midland town next
day. The door opened and a girl came in quickly, with a shawl over her head and a
basket on her arm.
“There’s a surprise for supper,” she said.
The old woman looked inside the basket. “Herrin’!” she said. “What did they
cost?”
“Tuppence apiece,” replied the girl lightly, as she hung up her shawl.
“They was cheap,” said the old woman as she fell to larding the frying-pan.
But all things considered, perhaps they were not so very cheap after all.
CHAPTER III
THE NAVY-THAT-FLIES[A]
The Royal Naval Air Service found itself “over the other side” about the time that
the shells of the British monitors began feeling for the hidden batteries of the
Boche behind the Belgian coast.
“I can’t see where they’re pitching,” said the Navy-that-Floats, referring to the
shells of the monitors bursting twelve miles away. “What about spotting for us, old
son?”
“That will I do,” replied the Navy-that-Flies. “And more also. But I shall have
to wear khaki, because it’s done, out here; by everybody apparently. Even the
newspaper reporters wear khaki. Also I must have the right machines and lots of
’em.”
“Wear anything you like,” replied the Navy-that-Floats, “as long as you can
help us to hit these shore batteries. Only—because you wear khaki and see life,
don’t forget you’re still the same old Navy as it was in the beginning, is now, and
ever shall be.”
The Navy-that-Flies added “Amen,” and said that it wouldn’t forget. It garbed
itself in khaki, but retained the ring and curl on the sleeve, and the naval cap (with
the eagle’s wings in place of the crown and anchor in the badge), plus a khaki cap-
cover. Wherever its squadrons were based they rigged a flagstaff and flew the
White Ensign at the peak. They erected wooden huts and painted them service
grey, labelling them “Mess-deck,” “Wardroom,” “Gunroom,” etc., as the case
might be.
They divided the flights into port and starboard watches, and solemnly asked
leave to “go ashore” for recreation. Those who strayed from the same stern paths of
discipline suffered the same punishments as the Navy-that-Floats. And at the
conclusion of each day’s work the wardroom dined, and drank to their King,
sitting, according to the custom and tradition of the naval service.
They filled in shell-holes and levelled the ground for aerodromes, they ran up
hangars and excavated dug-outs—whither they retired in a strong, silent rush (the
expression is theirs), when the apprehensive Boche attempted to curtail their
activity with bombs.
And by degrees the right machines came along. The Navy-that-Flies swung
itself into them critically, flung them about in the air three miles high, testing and
measuring their capabilities. Then they fought them, crashed them, improved on
them till they were righter still, and finally proceeded (to quote another of their
expressions) to “put the wind up Old Man Boche” in a way that helped the Navy-
that-Floats enormously.
But apart from spotting duties, which were necessarily intermittent, the
R.N.A.S. undertook a photographic reconnaissance of the entire Belgian coast from
Nieuport to the Dutch frontier. The work in progress at Ostend and Zeebrugge, the
activities of submarines and destroyers inside the basins; locks, quays, and gun-
emplacements, and the results of bombs dropped thereon the night before, were all
faithfully recorded by these aerial cameras. The negatives were developed and
printed, the resultant bird-pictures enlarged, studied through stereoscopic lenses,
and finally given to the monitors “for information and guidance.” Since it is not
given to everyone to recognise the entrance to a dug-out or a group of searchlights
as they appear from a height of 20,000 feet, the photographs were embellished with
explanatory notes for the benefit of anyone unaccustomed to such unfamiliar
aspects of creation.
The Germans claim to be a modest people. They were as busy as beavers, and
they resented these importunate photographers with all the fervour that springs
from true modesty. Their anti-aircraft guns plastered the intruders with bursting
shrapnel, and from every coast aerodrome Boche machines rose like a cloud of
angry hornets to give battle. Yet day after day fresh plates find their way to the
developing trays, and a comparison between the official reports of the flight—
couched in a laconic terseness of phrase that is good to read—and the amazing
results obtained gives perhaps the truest measure of the work performed by these
very gallant gentlemen.
Not a spadeful of earth can be turned over, nor a trowel of cement added to a
bastion along the coast, but a note appears a day or two later upon the long chart
which adorns the record office of this particular squadron. A crumpled escorting
machine may have come down out of the clouds, eddying like a withered leaf, to
crash somewhere behind the German line; there may be somewhere near the shore
a broken boy in goggles and leather lying amid the wreckage of his last flight. Such
is the price paid for a few more dots added in red ink to a couple of feet of chart.
But as long as the photographic machine returns with the camera intact, the price is
paid ungrudgingly.
The work of these photographic recorders, pilot and observer alike, differs from
all other forms of war flying. Their sole duty is to take photographs, not haphazard,
but of a given objective. This necessitates steering a perfectly steady course
regardless of all distractions such as bursting “Archies” and angry “Albatross”
fighters. They leave the fighting to their escorts, and their fate to Providence. The
observer, peering earthwards through his view-finder, steers the pilot by means of
reins until he sights the line on which the desired series of photographs are to be
taken: once over this, the pilot flies the machine on an undeviating course, and the
observer proceeds to take photographs. When all the plates have been exposed,
they turn round and return home with what remain of the escort. On occasions the
escort have vanished, either earthwards or in savage pursuit of resentful though
faint-hearted Boches; this is when the homing photographers’ moments are apt to
become crowded with incident.
One such adventure deserves to be recorded. It happened about 12,000 feet
above mother-earth: the official reports, typed in triplicate, covered some dozen
lines; the actual events, an equal number of minutes; but the story is one that
should live through eternity.
“While exposing six plates” (says the official report of this youthful Recording
Angel) “observed five H.A.’s cruising.” (“H.A.” stands for Hostile Aeroplane.)
“Not having seen escort since turning inland, pilot prepared to return. Enemy
separated, one taking up position above tail and one ahead. The other three glided
towards us on port side” (observe the Navy speaking), “firing as they came.
“The two diving machines fired over one hundred rounds, hitting pilot in
shoulder.” As a matter of sober fact, the bullet entered his shoulder from above and
behind, breaking his left collar bone, and emerged just above his heart, tearing a
jagged rent down his breast. Both his feet, furthermore, pierced by bullets, but the
observer was not concerned with petty detail.
“Observer held fire until H.A. diving on tail was within five yards.”
Here it might be mentioned that the machines were hurtling through space at a
speed in the region of one hundred miles an hour. The pilot of the “H.A.,” having
swooped to within speaking distance, pushed up his goggles and laughed
triumphantly as he took his sight for the shot that was to end the fight. But the
observer had his own idea of how the fight should end.
“Then shot one tray into pilot’s face,” he says, with curt relish, and watched him
stall, sideslip, and go spinning earthward in a trail of smoke.
He turned his attention to his own pilot. The British machine was barely under
control, but as the observer rose in his seat to investigate, the foremost gun fired,
and the aggressor ahead went out of control and dived nose-first in helpless spirals.
Suspecting that his mate was badly wounded in spite of this achievement, the
observer swung one leg over the side of the fuselage and climbed on to the wing—
figure for a minute the air pressure on his body during this gymnastic feat—until
he was beside the pilot. Faint and drenched with blood, the latter had nevertheless
got his machine back into complete control.
“Get back, you ass,” he said, through white lips, in response to inquiries as to
how he felt. The ass got back the way he came, and looked round for the remainder
of the “H.A.’s.” These, however, appeared to have lost stomach for further fighting,
and fled. The riddled machine returned home at one hundred knots, while the
observer, having nothing better to do, continued to take photographs. “The pilot,
though wounded, made a perfect landing.” Thus the report concludes.
The Navy-that-Flies had been in France some time before the Army heard very
much about its doings. This was not so much the fault of the Army as the outcome
of the taciturn silence in which the Navy-that-Flies set to work. It had been bidden
to observe the traditions of the silent Navy, and it observed them, forbearing even
to publish the number of Boche machines it accounted for day by day.
But there came a time when its light could no longer be hid under a bushel.
“Hullo,” said the generals and others concerned with the affairs of the entrenched
Army, speaking among themselves, “what about it?” They consulted the Army-
that-Flies.
Now the Army-that-Flies had been confronted in the early days of the war with
perhaps the toughest proposition that was ever faced by mortals of even their
imperturbable courage. In numerical inferiority to the enemy it had been called
upon to maintain a ceaseless photographic reconnaissance far behind the enemy’s
trenches; to spot for the guns of the Army along a suddenly extended front: to
“keep the wind up” the Boche so that for every ten of our machines that crossed the
German lines, barely one of his would dare to cross ours. This is called aerial
supremacy, and they established and maintained it with fewer and worse machines
than they care to talk about to-day.
“Of course we know all about these naval Johnnies,” said the Army-that-Flies.
“They’d steal grey paint from their dying grandmothers, and they fear nothing in
the heavens above, nor the earth beneath, nor in the waters under the earth. They
are complaining that things are getting a bit dull along the coast.... We might show
them a thing or two if they cared to join up with us for a while.”
“Let’s ask them,” said the Army.
So the Navy-that-Flies was invited “to co-operate with the Royal Flying Corps
on such portions of the line where its experience of escort work and offensive
patrols would prove of the greatest value.” Or words to that effect.
The Navy-that-Flies accepted the invitation with suppressed exultation, and
detailed certain squadrons of fighters. It admits having selected picked pilots,
because there was the credit of the old Navy to consider. Each squadron was
entrusted to the care of a seasoned veteran of fully twenty-five summers, and of the
flight leaders there was one that had even turned twenty-one. In short the Navy-
that-Flies was sending of its best; and its worst was very good indeed.
They flew away from the coast and the sea, and their motor transport rumbled
through the empty plains of France, till they closed upon the fringe of the
entrenched army. Here perched above the surrounding country on some plateau or
hill-side, with the ceaseless murmur of the guns in their ears, each of the squadrons
rigged its flagstaff and hoisted the White Ensign, set up the grey-painted huts and
the ship’s bell that divided the day into ship-watches, slung their hammocks, and
announced that they were ready to “co-operate” with anybody or anything.
The Army-that-Flies laughed at the ship’s bell and the rest of the naval
shibboleth, but it took the visitors to its heart. With hands deep in the pockets of its
“slacks” and pipe in mouth it came over and examined the fighting machines of the
Navy-that-Flies and the “doo-hickies” thereof, and it said things under its breath.
The Navy-that-Flies did not waste much time looking about it. One fire-eater
setting off to explore the country some thirty miles behind the German lines came
upon a school of “Quirks.” Quirks, it may be explained for the benefit of bipeds,
are young Boche aviators in an embryonic stage. From the convenient ambush of a
cloud he watched their antics for a while, as they flopped about above their
aerodrome; and then, descending like a thunderbolt, he tumbled three over,
scattered the remainder and returned to make his report. The squadron listened
gravely to the story and concluded that the Golden Age had dawned.
But sterner work lay ahead, and a fair sample of it is contained in the report of
another young gentleman who went scouting singlehanded over the German lines
what time the “gentlemen of England” were, if not abed, cracking the first of their
breakfast eggs.
He was attacked by two single-seated “Albatross” machines and a Halberstadt
fighter. Into the engine of the latter he emptied a tray of cartridges, with the result
that it immediately went spinning down; to make assurance doubly sure he fired
another fifty rounds into the whirling wreck as it fell.
By this time a veritable hornet’s nest appears to have risen about his ears; three
more “Albatross” machines whirred to the attack, and in his subsequent report he
notes with artistic enjoyment that the head of one pilot precisely filled the ring of
his sight. This eye for detail enabled him to recall the fact that he actually saw three
bullets strike the pilot’s head, with the not surprising result that the would-be-
avenger heeled over and sped to the ground.
By this time he had been driven down to a height of 200 feet above German-
occupied territory, and, having lost sight of the remainder of his aggressors, he
decided to return home at that height.
As was to be expected, his adventures were by no means terminated by this
decision. An astonished company of German cavalry drew rein and peppered him
with rifle-shots as he whisked over the tops of their lances. Five minutes later
another “Albatross” attacked him.
He rocked the machine in giddy sweeps until within fifty yards of his opponent,
and side-looped over him (this, remember, at 200 feet from the ground), fired a
short burst and drove the Hun off for a moment while he regained equilibrium.
Then once more the enemy swooped upon him.
From this point onwards the reader may be warned against vertigo. The pilot’s
own version, the bald official report of the affair, requires no embellishment or
comment, though the latter is not easy to suppress.
“These operations,” he states, “were repeated several times with a slight
variation in the way I looped over him (flying against a head wind). When he was
about 150 yards behind me, I looped straight over him, and coming out of the loop
dived at him and fired a good long burst. I saw nearly all the bullets go into the
pilot’s back, just on the edge of the cockpit. He immediately dived straight into the
ground.
“I then went over the German trenches filled with soldiers, and was fired on by
machine guns, rifles, and small field guns, in or out” (Ye Gods and Little Fishes!)
“of range. There were many shells bursting in and about the German trenches.”
The report concluded with estimates of the strength of various bodies of
infantry and cavalry, movements of convoy and artillery noticed during the
intervals between aerial somersaults. The pilot landed at the first aerodrome he saw
—adding, in explanation of such an irregular proceeding, that his machine was
badly shot about.
The squadrons co-operating with the R.F.C. commenced by faithfully recording
all aerial combats in which their machines were engaged. But after a while such
events became too commonplace to chronicle. They fought from dawn to dusk,
generally a day’s journey for a horse behind the German lines. They fought at
altitudes at which in spring a thermometer registered 50° of frost, returning with
petrol tanks frozen, and hands and feet and ears swollen by frost-nip. One squadron
had a hundred decisive fights in a month (omitting skirmishes), and accounted for
twenty-five Boche machines. Its log (unofficially termed “Game-book”) contained
such entries as the following: “Four machines went up: managed to bag five Huns
before breakfast.”
For the first time in their lives the pilots got all the fighting they wanted, and
revelled in it gluttonously. They grew fine-drawn, with the accentuated brilliancy
of eye common to men in perfect condition living at the highest tension. They met
Winged Death hourly in the blue loneliness above the clouds; the rustle of his sable
wings became a sound familiar as the drone of their own engines, so that all terror
of the Destroyer passed out of their souls—if indeed it had ever entered there.
And Death in his turn grew merciful, amazed. At least this is the only
explanation to offer for certain tales that are told along the Front, where the White
Ensign flies.
But hear for yourselves and judge.
A Naval pilot—a Canadian, by the way—attacked a single-seater “Albatross”
scout at 8,000 feet above the German lines. He disposed of him after a short
engagement, and was then attacked by seven others who drove him down to 3,000
feet and shot his machine to pieces. He plunged to the ground and crashed amid the
wreck of his machine a couple of hundred yards behind the Canadian lines,
breaking a leg and dislocating a shoulder. A furious bombardment from German
heavy artillery was in progress at the time, and he crawled into a shell-hole, where
he remained from 9 a.m. until 4 p.m. Fire then having slackened, a party from the
trenches went in search of his body with a view to burying it, and found him
conscious and cheerful, though very thirsty.
The Navy-that-Flies is witness that I lie not.
As far as bombing operations are concerned, the Navy-that-Flies confines its
attentions principally to the German bases along the Belgian coast, and any lurking
submarines or vagrant destroyers observed in the vicinity. Bombing is carried out
by both aeroplanes and seaplanes, and differs from other forms of war flying in that
it is principally performed at night.
The function of the bombing machine is to reach its given objective in as short a
time as possible, without provoking more “scraps” on the way than are inevitable,
to “deliver the goods,” and, if not brought down by anti-aircraft fire, to return with
all speed. They are not primarily fighters, and when laden with bombs are not
theoretically a match for a hostile fighting machine with unfettered manœuvring
powers.
Engine-trouble or loss of stability over enemy territory means almost infallible
capture or death for the pilot of a bombing aeroplane. Yet in cases of disablement,
rather than come down on the ground and suffer themselves or their machine to be
taken prisoner, it is their gallant tradition to try to struggle out to sea. Here they
stand about as much chance of life as a pheasant winged above a lake, but the
machine sinks before German hands can touch it.
Now it happened that on one such occasion the descent into the sea of a
bombing machine was observed by two French flying boats which were out on
patrol. The distressed machine was still within range of the shore batteries, and the
Boches, smarting under the effect of the bombs she had succeeded in dropping,
were retaliating in the most approved Germanic manner by plastering the helpless
machine with shrapnel as she slowly sank.
The two French flying boats sped to the rescue and alighted in the water beside
the wrecked British machine. One embarked the observer, who was wounded, and,
in spite of redoubled fire from the shore, succeeded in returning safely. The other
French flying-boat actually embarked the remaining occupants of the bombing
machine, but was hit as it rose from the water and fell disabled. The French pilot,
seeing a Boche seaplane approaching, and a bevy of small craft in-shore coming
out against them, scribbled a message to say that his venture had failed; he found
time to add, however, with true Gallic dauntlessness of spirit, “Vive la France!”
This missive he fastened to the leg of his carrier pigeon, and succeeded in releasing
it before rescuers and rescued were taken prisoners.
From time to time curt official announcements of successful bomb-raids upon
German destroyer and submarine bases appear in the press. It may be that the
Naval honours or casualties lists are swelled thereby. But no one who has not stood
in the wind that blows across the bombers’ aerodrome at night, in those last tense
moments before the start, can form any idea of the conditions under which these
grim laurels are earned.
One by one the leather-clad pilots conclude their final survey and climb up into
their machines. They adjust goggles and gloves: there is a warning “Stand clear!”
and the darkness fills with roaring sound as No. 1 starts his engine. For a moment
longer he sits in the utter isolation of darkness and the deafening noise of his own
engine. No further sounds can reach him; not another order nor the valedictory
“Good luck!” from those whose lot it is to only stand and wait. He settles himself
comfortably and fingers the familiar levers and throttle; then with a jerk the
bomber starts along the uneven ground, gathers way, and rising, speeds droning
into the darkness like a gigantic cockchafer. A moment later No. 2 follows, then
another, and another. The night swallows them, and the sound of their engines dies
away.
A couple of hours later in one of the grey-painted huts that fringe the aerodrome
a telephone bell jangles. The squadron commander picks up the receiver and holds
converse with a tiny metallic voice that sounds very far away; the conversation
ends, he puts on his cap and goes out into the darkness; a few minutes later a
sudden row of lights across the aerodrome makes bright pin-pricks in the darkness.
From far away in the air comes the hum of an engine growing momentarily louder.
It grows louder and clearer as the homing machine circles overhead and finally
comes to earth with a rushing wind and the scramble of men’s feet invisible.
The pilot climbs stiffly out of his seat, pushing up his goggles, and puckers his
eyes in the light of the lanterns as he fumbles for his cigarette case. “Got ’em,” he
says laconically. “Seaplane sheds on the mole. Time for another trip?”
There is time, it appears. He drinks hot coffee while the armourers snap a fresh
supply of bombs into the holders and test the release gear. He answers questions
curtly and his replies are very much to the point.
Their “Archies” are shooting well, and they’ve got a lot more searchlights at
work than they had last time. Rather warm work while it lasted. He thinks No. 1
was hit and brought down in flames. No. 2 seemed to have engine trouble this side
of our lines on the way back. No. 3 ought to be along soon. And while he gulps his
coffee and grunts monosyllables there is a whirring overhead and No. 3 returns,
loudly demanding a fresh supply of bombs with which to put an artistic finish to a
row of blazing oil-tanks.
They climb into their machines again and lean back resting, while the finishing
touches (which sometimes come between life and death) are put to the machines
and their deadly freight. Then once more they soar up into the night.
Dawn is breaking when No. 4 returns, tired-eyed, and more monosyllabic than
ever. It came off all right, but No. 3 had seemed to lose control and slid down the
beam of a searchlight with shells and balls of red fire (some new stunt, he
supposed) bursting all about her. However, she got her bombs off first, and touched
up something that sent a flame 200 feet into the air. He himself bombed a group of
searchlights that were annoying him, and some trucks in a railway siding. The
speaker has an ugly shrapnel wound in the thigh and observes with grave humour
that his boots are full of blood—this is a Navy joke, by the way. Also that he could
do with a drink.
But it came off all right.
Now the seaplanes, who undertake much the same sort of job, keep pigs, and
contemplate their stern mission with an extinguishable and fathomless sense of
humour. This may be accounted for by the fact that in life and death they are more
in touch with the native element of the Navy-that-Floats and share much of its
light-heartedness in consequence.
Aerial gymnastics are not in their line. They fight when they must, and the
straightest shot wins. If hit, unless hopelessly out of control, they take to the water
like a wounded duck. If the damage is beyond temporary repair they sit on the
surface and pray for the dawn and a tow from a friendly destroyer.
No aerial adventure is ever recounted (and the array of medal ribbons round
their mess table is witness to the quality of these blindfold flights) without its
humorous aspect well-nigh obliterating all else. One who fought a Zeppelin single-
handed with a Webley-Scott pistol and imprecations found himself immortalised
only in the pages of a magazine of Puck-like humour they publish (Fate and funds
permitting) monthly. Another, disabled on the water off an enemy’s port, succeeded
in getting his engine going as the crew of an armed trawler were leaning over the
bows with boat-hooks to secure him. He rose from the water beneath their
outstretched hands, and recalled with breathless merriment nothing but the
astonishment on their Teutonic faces. A third, similarly disabled, was approached
on the surface by a German submarine. He raked her deck with his Lewis gun and
kept her at bay—by the simple expedient of picking off every head that appeared
above her conning-tower—until she wearied of the sport and withdrew. From a
seaplane point of view it was a pretty jest.
At the conclusion of a day’s aerial fighting on the Somme front a certain officer
of the Navy-that-Flies was asked how he felt about it.
“Wa-al ...” he drawled, and paused, groping in his mind for metaphor. “It’s jest
like stealing candy from a kid.”
Making all allowances for poetic licence, this is a very fair illustration of the
spirit in which the Navy-that-Flies went about the business. On the other hand
there were a few who took a graver view of their responsibilities.
Among the possessions of one of the naval squadrons co-operating with the
Army-that-Flies along the front is a foolscap manuscript notebook bearing the
superscription Notes on Aerial Fighting. The youthful author of these notes will
never handle either pen or “joy-stick” again, but he has left behind him a document
that is, in its way, one of the epics of war literature. It has since been printed (in
expurgated form), and has doubtless found its way into textbooks and treatises on
the subject. But to be appreciated to the full it should be read in the original round,
rather boyish handwriting, within hearing of the continuous murmur of the British
guns and the drone of a scouting fighter passing overhead.
It contains ten commandments, which, for a variety of reasons, need not be
recapitulated here. But the introduction epitomises the spirit of them all:
“The man who gets most Huns in his lifetime is the man who observes these
commandments and fights with his head. The others either get killed or get nerves
in a very short time and the country does not get the full benefit of having trained
them.”
The commandments conclude with the following exhortation: “A very pleasant
(sic) help in time of trouble is to put yourself in the enemy’s place and view the
situation from his point of view. If you feel frightened before an attack, just think
how frightened he must be!”
The Navy-that-Floats possesses for its “pleasant help” an awesome volume of
some 946 pages (not counting Addenda), entitled The King’s Regulations and
Admiralty Instructions. Yet in all its pages there is not one clause which can
compare with this brave sentence: for this is youth speaking to youth, for the
guidance and comfort of his soul.
Now in one of the squadrons of the Navy-that-Flies there are three flight
leaders, and the sum total of their ages is fifty-nine. The youngest, whatever his
birth certificate may testify, looks something under sixteen. Of him it is related that
in his early youth, having brought down a hostile machine within the British lines
and captured the two occupants (with Iron Crosses complete), he approached a
certain general, demanding transport for his prisoners—covering them the while
with an automatic pistol.
“Transport?” said the general. “Where d’you want to take them?”
“To my squadron headquarters,” was the grave reply. “I’d like to keep ’em for a
bit. I’d like the others to see ’em.”
“Damn it,” replied the General, “they ain’t canaries. Certainly not. Send ’em to
the cages with the rest of the prisoners.”
The victor flew sorrowfully homewards, and on arrival gave it as his opinion
that professional jealousy was the ruination of the Junior Service....
They are not given to talking over-much of their achievements in the hearing of
a stranger within their gates. The second youngest of the trio admitted,
contemplating his cow-hide boots, to have “done-in” twelve hostile machines in
single combat—and lapsed into agonised silence.
“Of course,” said the third, coming to the rescue of a comrade in palpable
distress, “N., the star Frenchman, is the fellow to talk if you want to hear some
good yarns.” The speaker had the grave, sweet face of a mediæval knight, and the
owner of the cow-hide boots shot him a swift glance of gratitude.
“He’s done-in fifty Huns,” he confirmed, nodding.
It was on the following day, as it happened, that Fate introduced the Frenchman
to the Stranger within the Gates of the Navy-that-Flies. The flying man landed on
one of the aerodromes of the Navy-that-Flies, a florid-faced young man, chubby
and blue-eyed. The squadron strolled out to greet him with ready hospitality and
hero-worship.
“Bon jour, N.,” said the squadron commander. “How goes it?”
The famous French fighting pilot swung himself out of his machine and pulled
off his gauntlet. He wore, in addition to the regulation flying helmet, a bright-blue
muffler wound with many turns round the lower part of his face, and a soiled
aquascutum with a row of medal-ribbons reaching half-way across his breast. The
wind fluttered its skirts, disclosing a pair of tight red breeches above top-boots of a
light yellow. As an additional protection against the cold his feet were encased in
fur moccasins. He greeted the Navy-that-Flies in rapid French and threw their
ranks into some disorder.
“Translate, George,” said the squadron commander.
“He says he’s on sick leave,” explained one of the hosts. “He’s just flying to
keep his eye in. He scuppered five Boches last week.”
“Si,” said the Frenchman, nodding, and held up his hand with outstretched
fingers, “Cinq!”
“Good on you, old sport,” said the squadron commander. They shook hands
again, and the remainder clustered rather curiously round the sinister machine with
the black skull and cross-bones adorning its fusilage.
“Makes one sort of sorry for the Hun, doesn’t it?” said one musingly.
“George,” said another, “ask him what that doo-hickie on the muzzle of his
gun’s for.” He indicated a detail on the mounting.
The Frenchman explained at some length, and the interpreter interpreted.
“Bon!” said the squadron commander.
“Oui,” said the Frenchman, “tres bon! You ’ave not eet—cette—comment dites
vous?—doo-hickie? No?”
“No,” was the reply, “mais nous blooming well allons——”
The Frenchman presently climbed back into his machine and took his departure.
The squadron commander summoned his chief armourer, and for a while deep
called to deep.
“He’s a red-hot lad, that Frenchman,” said the squadron commander, when the
chief armourer had gone. “I fancy he only came down to let us see that doo-hickie
of his on his gun. You ought to hear some of his yarns, though.”
The Stranger within the Gates of the Navy-that-Flies gazed after the aerial
speck against the blue of heaven, and his soul was glad within him, because it was
all the purest Navy.
“That’s all right,” he said. “But what I should like to know is, what the deuce is
a doo-hickie?”
“A doo-hickie?” replied the squadron commander. “A doo-hickie? H’m’m.
George, how would you describe a doo-hickie?”
The officer appealed to puffed his pipe in silence for a moment. “Well,” he said
at length, “you know more or less what a gadget’s like?”
“Yes.”
“And a gilguy?”
“Yes.”
“Well, a doo-hickie is something like that, only smaller as a rule.”
There was a silence. Then the squadron commander leaned forward and flicked
a speck of fluff off the shoulder of the Stranger within their Gates.
“There you are!” he exclaimed triumphantly—“that’s a doo-hickie!”
“Have a drink, anyway,” said the officer who answered to the name of George,
soothingly.
The Stranger within the Gates of the Navy-that-Flies had the drink, and from
then onwards forbore to ask any more questions.
But he still sometimes wonders what the functions of a doo-hickie might be.
CHAPTER IV
“LEST WE FORGET”
I. H.M.S. “Shark”
H.M.S. Shark, under the command of Commander Loftus W. Jones, went into
action about 5.45 p.m. on May 31st, 1916 with a complement of ninety-one
officers and men; of that number only six saw June 1st dawn.
In spite of the soul-shaking experience through which they passed, these six
men have remembered sufficient details of the action to enable the following
record to be pieced together. Many stirring acts of gallantry and self-sacrifice, and
much of interest to the relatives and friends of those who were lost, must inevitably
be lacking from this narrative. But the evidence shows such supreme human
courage and devotion to duty in the face of death, that, incomplete as it is, the story
remains one of the most glorious in the annals of the Navy.
At two o’clock on the afternoon of May 31st the Shark and three other
destroyers, Acasta, Ophelia, and Christopher, were acting as a submarine screen to
the Third Battle Cruiser Squadron, with the light cruisers Chester and Canterbury
in company. The force was steaming on a southerly course in advance of the
British Battle Fleet, which was engaged in one of its periodical sweeps of the North
Sea.
This advance squadron was under the command of Rear-Admiral the Hon.
Horace A. L. Hood, C.B., M.V.O., D.S.O., flying his flag in Invincible.
The main Battle Cruiser Fleet and the Fifth Battle Squadron were considerably
farther to the southward, and at 2.20 p.m. the light cruisers attached to this force
signalled by wireless the first intimation that the enemy’s fleet was at sea.
Subsequent reports confirmed this, and acting on the information contained in
these intercepted messages, Rear-Admiral Hood ordered the ship’s companies to
“Action Stations,” and shaped course to intercept the advancing enemy.
At 3.48 p.m. the Battle Cruiser Fleet and the Fifth Battle Squadron engaged the
German Main Fleet and turned north with the object of drawing the enemy towards
the British Battle Fleet. It must be remembered that at this point the enemy was
presumably in complete ignorance of the approach of the British Main Fleet. The
weather was hazy, with very little wind and patches of mist that reduced the
visibility to an extent that varied from one to eight miles.
At 4.4 p.m. Rear-Admiral Hood received orders from Admiral Sir John Jellicoe,
Commander-in-Chief, to proceed at full speed with his squadron and reinforce the
Battle Cruiser Fleet; the Third Battle Cruiser Squadron altered course as necessary,
and an hour and a half later the first sounds of firing reached them out of the mists
ahead.
The first faint intermittent murmur of sound increased momentarily as the two
forces converged, and at 5.40 p.m. the haze on the starboard bow was pierced by
flashes of gunfire; a few minutes later a force of German light cruisers and
destroyers became visible, heavily engaged with an unseen opponent to the
westward.
Fire was immediately opened and Rear-Admiral Hood turned to starboard,
bringing the enemy on to the port bow of his squadron. Three light cruisers, a
flotilla leader, and ten destroyers were now visible, the latter apparently turning to
launch a torpedo attack upon the Third Battle Cruiser Squadron. The four
destroyers who had hitherto been disposed in two subdivisions, one on each bow of
the Invincible, were thereupon ordered to attack the enemy. Led by Commander
Loftus Jones in the Shark, the division swung round, and hurled itself at the
German force, opening fire with every gun that would bear.
In the meanwhile the enemy opened a heavy though ill-directed fire on the
battle cruisers. A large proportion of the salvos were falling short, and the British
destroyers had in consequence to advance through a barrage of fire which
surrounded them on all sides with columns of water and bursting shell.
In spite of their numerical superiority, the German destroyers turned away in the
face of this determined onslaught, and Commander Loftus Jones, satisfied that the
intended torpedo attack on Rear-Admiral Hood’s squadron had been frustrated, and
having fired two of his three torpedoes, turned sixteen points to regain his position
on the bow of the Invincible. The remaining three destroyers followed in his wake.
Three German battle cruisers had then appeared out of the mist in support of the
enemy light cruisers, and the gallant division, with Shark at their head, turned
under a concentrated deluge of shells from the entire German force.
A fragment of a projectile struck the Shark’s wheel, shattering it, and wounding
the coxswain, Petty Officer Griffin, on the right hand. The captain immediately
ordered the after wheel to be manned and followed the coxswain down the ladder
to the shell-torn upper-deck to con the ship from aft. The yeoman of signals, Petty
Officer Banham, who up to this point had been the third occupant of the bridge,
hurried after the captain.
The enemy were now using shrapnel, and the captain was wounded in the thigh
and face as he reached the bottom of the ladder. He stumbled aft, endeavouring to
staunch the flow of blood with his hands, to find on reaching the engine-room
hatchway that a shell had burst inside the engine-room, and the main engines and
steering gear were completely disabled. The coxswain had been struck at the same
time as the captain, and dropped insensible from a wound in the head. The
foremost gun, under the command of Sub-Lieutenant Vance, had been blown away,
and only one survivor of its crew lay badly wounded amid the wreckage.
The Shark was then lying with disabled engines helpless under a heavy fire, and
Lieutenant-Commander John O. Barren, who commanded the Acasta, and had been
second in the line, gallantly brought his destroyer between the Shark and the
enemy’s fire, and signalled to ask if he could be of any assistance. The captain of
the Shark was then aft, cheering and encouraging the crews of the midship and
after guns. The yeoman of signals, who remained at his side, read the signal and
reported it to the captain, who replied, “No. Tell him to look after himself and not
get sunk over us.”
The yeoman of signals accordingly semaphored Commander Jones’s last signal
to the division under his orders, and the Acasta followed in the wake of the other
two boats which were rejoining the battle cruisers.
It is probable that at this juncture Rear-Admiral Hood sighted the British Battle
Cruiser Fleet, which he had been ordered to reinforce, and proceeded to carry out
his orders. The Third Battle Cruiser Squadron vanished into the mist, and the
enemy closed in upon the Shark, which lay rolling helplessly in the swell, blazing
defiance from her after and midship guns.
The after gun was almost immediately put out of action and its crew killed and
wounded. Amid a hail of shrapnel bullets and flying splinters the spare torpedo was
hoisted off the rack, and, under the directions of the captain, was being launched
into the tube, when it was struck by a shell and burst with a violent explosion,
causing heavy casualties.
Only one gun, the midship one, now remained in action. The ship was settling
down by the bows and every moment she shuddered with the impact of a fresh hit.
The riven upper-deck was a shambles, and the dead, mingled with shattered
wreckage, were blown hither and thither by the blast of exploding shell. Projectiles,
pitching short, flung great columns of water into the air, or passed screaming
overhead; the upper-works were riddled by splinters from bursting salvos.
One by one the wounded crawled brokenly into the lee of the casings and
funnels in pitiful attempts to find shelter; among them knelt the devoted figure of
the surgeon (Surgeon-Probationer Robert Walker, R.N.V.R.), endeavouring single-
handed to cope with his gallant, hopeless task. When last seen he was bandaging a
man who had lost a hand when the torpedo exploded. He was then himself severely
wounded, and was apparently shortly afterwards killed.

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Lactic Acid Bacteria Bioengineering

and Industrial Applications Wei Chen

Lactic Acid Bacteria: Methods and Protocols Makoto

Lactic acid bacteria : microbiological and functional

Lactic Acid Bacteria-A Functional Approach 1st Edition

Acetic Acid Bacteria: Fundamentals and Food

Industrial Revolution 4.0, Tech Giants, and Digitized

Studies on Green Synthetic Reactions Based on Formic

Current Developments in Biotechnology and

Current Developments in Biotechnology and

Lactic Acid Bacteria

ISBN 978-981-13-7282-7 ISBN 978-981-13-7283-4 (eBook)

1 Lactic Acid Bacteria and γ-Aminobutyric Acid and Diacetyl�������������� 1

Shunhe Wang, Pei Chen, and Hui Dang

1.1 Lactic Acid Bacteria and γ-Aminobutyric Acid

1.1.1 Introduction of γ-Aminobutyric Acid

γ-Aminobutyric acid (GABA), or γ-ammonia butyric acid, is a kind of nonprotein

© Springer Nature Singapore Pte Ltd. and Science Press 2019 1

1.1.1.2 General Physicochemical Property

1.1.2 GABA Production of Lactic Acid Bacteria

The production of GABA by microbial fermentation is to use the glutamate decar-

1.1.3 Physiological Function of γ-Aminobutyric Acid

1.1.3.1 Delaying the Senescence of Nerve Cells

1.1.3.2 Lower Blood Pressure

1.1.3.3 Effects on Physiological Reproduction

1.1.3.4 Treatment of Mental Illness

1.1.4 Application of γ-Aminobutyric Acid

The development of GABA-enriched foods began in 1986. GABA-enriched teas

1.2 Lactic Acid Bacteria and Diacetyl

1.2.1 Introduction of Diacetyl

Diacetyl, or butanedione (chemical name, 2,3-butanedione), is a yellow or light

that lower pH enhanced the bacteriostatic activity of diacetyl (Meng Xiangchen

1.2.2 Diacetyl Production of Lactic Acid Bacteria

Many microorganisms could produce diacetyl, such as Streptococcus, Leuconstoc,

1.2.3  roduction Way and Metabolic Regulation of Diacetyl

1.2.3.1.1 Production of Diacetyl by Citric Acid Metabolic Pathways

Fig. 1.1 Citric acid metabolic pathways in Lactobacillus

1.2.3.1.2 Production of Diacetyl by Glucose Metabolic Pathways

Fig. 1.2 Synthesis pathways of acetaldehyde and diacetyl in Lactobacillus

by its synthetase. And diacetyl was generated by nonenzymatic natural oxidative

1.2.3.2 Metabolic Regulation of Diacetyl in Lactic Acid Bacteria

1.2.3.2.1 Regulation of Culture Conditions

Production and stability of diacetyl depended on the growing environment of lactic

1.2.3.2.2 Regulation of α-Acetolactic Acid

The metabolism of pyruvate and citrate in Lactococcus and Leuconostoc confirmed

The inactivation of LDH led to excessive accumulation of pyruvate. In theory,

1.2.3.2.3 Regulation of Mixed Acid

Under different fermentation conditions, researchers studied the production of

Generally, Lactobacillus was the homolactic fermentation, and metabolic changes

Fig. 1.3 Mixed acid fermentation of pyruvate

1.2.4  trategy for Increasing Production of Diacetyl

1.2.4.2  pplication of Genetic Engineering and Metabolic Engineering

1.2.5 Application of Diacetyl in Foods

Diacetyl inhibited many microorganisms, including spoilage and pathogenic bacte-

Ai M (1984) Oxidation of methyl ethyl ketone to diacetyl on V2O5-P2O5 catalysts. J Catal

Wei Chen, Bo Yang, and Jianxin Zhao

2.1.1 Conjugated Linoleic Acid

W. Chen (*) · B. Yang · J. Zhao

© Springer Nature Singapore Pte Ltd. and Science Press 2019 21

1 Lactic Acid Bacteria and γ-Aminobutyric Acid and Diacetyl�� 1

1.1 Lactic Acid Bacteria and γ-Aminobutyric Acid

1.1.1 Introduction of γ-Aminobutyric Acid

1.1.1.2 General Physicochemical Property

1.1.2 GABA Production of Lactic Acid Bacteria

1.1.3 Physiological Function of γ-Aminobutyric Acid

1.1.3.1 Delaying the Senescence of Nerve Cells

1.1.3.2 Lower Blood Pressure

1.1.3.3 Effects on Physiological Reproduction

1.1.3.4 Treatment of Mental Illness

1.1.4 Application of γ-Aminobutyric Acid

1.2 Lactic Acid Bacteria and Diacetyl

1.2.1 Introduction of Diacetyl

1.2.2 Diacetyl Production of Lactic Acid Bacteria

1.2.3 roduction Way and Metabolic Regulation of Diacetyl

1.2.3.2 Metabolic Regulation of Diacetyl in Lactic Acid Bacteria

1.2.4 trategy for Increasing Production of Diacetyl

1.2.4.2 pplication of Genetic Engineering and Metabolic Engineering

1.2.5 Application of Diacetyl in Foods

2.1.1 Conjugated Linoleic Acid

2.1.2 Conjugated Linolenic Acid

2.1.3 Other Conjugated Fatty Acid

2.2 actic Acid Bacteria with Conjugated Fatty Acid

2.2.1 Conjugated Fatty Acid Production in Lactobacilli

2.2.1.1 Lactobacillus plantarum