| 1. Present situation
of the probiotics' research |
| "Probiotics" generally includes bacteria,
cyanobacteria, micro algae fungi, etc. Some Chinese researchers translate
it into English as "Normal micro biota" or "Effective micro
biota"; it includes Photosynthetic bacteria, Lactobacillus, Actinomycetes,
Nitrobacteria, Denitrifying bacteria, Bifidobacterium, yeast, etc.
Usually, it does not include micro algae. In English literature, probiotic
bacteria are generally called the bacteria which can improve the water quality
of aquaculture, and (or) inhibit the pathogens in water there by increasing
production. "Probiotics", "Probiont", "Probiotic
bacteria" or "Beneficial bacteria" are the terms synonymously
used for probiotic bacteria. |
| The theory of ecological prevention and cure
in controlling the insect pest of terrestrial higher grade animals and plants
has been in practice for long time, and has achieved remarkable success.
The use of beneficial digestive bacteria in human and animal nutrition is
well documented. Lactobacillus acidophilus
is used commonly to control and prevent infections by pathogenic microorganisms
in the intestinal tract of many terrestrial animals. Recently,
the biocontrolling theory has been applied to aquaculture. Many researchers
attempt to use some kind of probiotics in aquaculture water to regulate
the micro flora of aquaculture water, control pathogenic microorganisms,
to enhance decomposition of the undesirable organic substances in aquaculture
water, and improve ecological environment of aquaculture. In addition, the
use of probiotics can increase the population of food organisms, improve
the nutrition level of aquacultural animals and improve immunity of cultured
animals to pathogenic microorganisms. In addition, the use of antibiotics
and chemicals can be reduced and frequent outbreaks of diseases can be prevented. |
| Nogami and Maeda (1992) isolated a bacteria strain
from a crustacean culture pond. The bacterial strain was found to improve
the growth of crab (Portunus trituberculatus) larvae and repress
the growth of other pathogenic bacteria, especially Vibrio spp., but
would not kill or inhibit useful micro algae in sea water when it was added
into the culture water. Among the bacteria population present in the culture
water of the crab larvae, the numbers of Vibrio spp. and pigment
bacteria decreased or even became undetectable when the bacteria was added
into culture water. The production and survival rate of crab larvae were
greatly increased by the addition of the probiotic bacteria into the culture
water. They also suggested that the bacterium might improve the physiological
state of the crab larvae by serving as a nutrient source during its growth.
This bacterium may have a good effect in the crab larval culture as a biocontrolling
agent in the future. |
| Austin et al (1992) reported a kind of
micro algae (Tetraselmis suecica), which can inhibit pathogenic
bacteria of fish. Teraselmis suecica was observed to inhibit Aeromonos
hydrophila, A. salmonicida, Serrstia liquefaciens,
Vibrio anguillaram, V. salmonicida and Yersnia ruckeri
type I. When used as a food supplement, the algal cells inhibited laboratory-induced
infection in Atlantic salmon. When used therapeutically, the algal cells
and their extracts reduced mortalities caused by A. salmonicida,
Ser. liquefaciens, V. anguillaram, V. salmonicida
and Yersnia ruckeri type I. They suggested that there may be some
bioactive compounds in the algal cells, and there appears to be a significant
role for Tetraselmis in the control of fish diseases. |
| Smith and Davey (1993) reported that a fluorescent
strain pseudomonad bacteria can competitively inhibit the growth of fish
pathogen A. salmonicida. Their results show that
the fluorescent pseudomonad is capable of inhibiting the growth of A.
salmonicida in culture media and that this inhibition is probably due
to competition for free iron. In a challenge test of the Atlantic
salmon by A. salmonicida, a statistically significant reduction
in the frequency of stress-induced infection in the group of fish bathed
in the bacterium fluorescent pseudomonad compared to the control group was
observed. |
| Austin et al (1995) reported a
probiotic strain of Vibrio alginolyticus, which did not cause any
harmful effect in salmonids. By using the cross-streaking method, the probiont
was observed to inhibit the fish pathogens. When the freeze-dried
culture supernatant was added to the pathogenic bacteria such as V.
ordalii, V. anguillarum, A. salmonicida and Y.
ruckeri, showed a rapid or steady decline in the number of culturable
cells, compared to the controls. Their results indicated that application
of the probiont to Atlantic salmon culture led to a reduction in mortalities
when challenged with A. salmonicida and to a lesser extent V.
anguillarum and V. ordalii. The observation with this probiotic
Vibrio is encouraging, and it appears that there is tremendous
potential for the use of such probiotics in aquaculture as part of a disease
control strategy. |
| Maeda and Nagami (1989) reported some aspects
of the biocontrolling method in aquaculture. In their study bacterial strains
possessing vibrio static activity which improved the growth of prawn and
crab larvae were observed. By applying these bacteria in aquaculture, a
biological equilibrium between competing beneficial and deleterious microorganisms
was produced, and results show that the population of Vibrio spp.,
which frequently causes large scale damage to the larval production, was
decreased. Survival rate of the crustacean larvae in these experiments showed
much higher than those without the addition of bacterial strains. They hope
that addition of these strains of bacteria will repress the growth of Vibrio
spp., fungi and other pathogenic microorganisms. Their data suggest
that controlling the aquaculture ecosystem using bacteria and protozoa is
quite possible and if this system is adopted, it will maintain the aquaculture
environment in better condition, which will increase the production of fish
and crustaceans. |
| Garriques and Arevalo (1995) reported that the
use of V. alginolyticus as a probiotic agent may increase survival
and growth in P. vannamei postlarvae by competitively excluding
potential pathogenic bacteria, and can effectively reduce or eliminate the
need for antibiotic prophylaxis in intensive larvae culture system. They
believe that in nature a very small percentage of Vibrio sp. is
truly pathogenic, and the addition of potentially pathogenic bacteria to
aquaculture system through water, algae, and/or Artemia was recognized.
In their study, the addition of the bacteria V. alginolyticus as
a probiotic to mass larvae culture tanks resulted in increased survival
rates and growth over the controls and the antibiotic prophylaxes. |
| Jiravanichpaisal and Chuaychuwong et al (1997)
reported the use of Lactobacillus sp. as the probiotic
bacteria in the giant tiger shrimp (P. monodon Fabricius).
They designed to investigate an effective treatment of Lactobacillus
sp. against vibriosis and white spot diseases in P. monodon.
They investigated the growth of some probiotic bacteria, and their survival
in the 20 ppt sea water for at least 7 days. Inhibiting activity of two
Lactobacillus sp. against Vibrio sp., E. coli,
Staphylococcus sp. and Bacillus subtilis was determined.
Direkbusarakom and Yoshimizu et al (1997) reported Vibrio spp.
which dominate in shrimp hatchery against some fish pathogens. Two isolates
of Vibrio spp. which are the dominant composition of the flora
in shrimp hatchery, were studied for antiviral activity against infectious
haematopoietic necrosis virus (IHNV) and Oncorhynchus masou virus (OMV).
Both strains of bacteria showed the antiviral activities against IHNV and
OMV by reducing the number of plaque. Their results demonstrate the possibility
of using the Vibrio flora against the pathogenic viruses in shrimp
culture. |
| Sugita and Shibuga (1996) reported the antibacterial
abilities of intestinal bacteria in freshwater cultured fish. They isolated
bacteria from the intestine of 7 kinds of freshwater cultured fish, and
investigated the antibacterial abilities of these bacteria to 18 fish or
human common pathogenic bacteria. Their results indicated that the
bacteria isolated from intestine of 7 kinds of freshwater cultured fish
possess the antibacterial abilities, and the presence of the intestinal
bacteria can protect the fish against the infection by pathogenic bacteria. |
| Maeda and Liao (1992) reported on the effect
of bacterial strains obtained from soil extracts on the growth of prawn
larvae of P. monodon. Higher survival and molt rates of
prawn larvae were observed in the experiment treated with soil extract,
and the bacterial strain which promoted the growth of prawn larvae was isolated.
They have assumed that if a specific bacterium is cultured and added to
the prawn ecosystem to the level of 10 million cell/ml, other bacteria may
hardly inhibit the same biotype because of protozoan activity which shall
be one of the way to biologically control the aquaculture water biotype
and ecosystem. |
| Maeda and Nogami et al (1992) have reported the
utility of microbial food assemblages in culturing a crab, Portunus
trituberculatus. Assemblages of microorganisms were produced by adding
several nutrients, urea, glucose and potassium phosphate, to natural seawater
with gentle aeration in which bacteria and yeast were prevailing. When these
cultured microbes were added to sea water where crab larvae of Portunus
trituberculatus were reared, bacteria numbers decreased very rapidly,
followed by the decrease in flagellated protozoa and diatoms. Their results
suggest that the crab larvae fed on these microorganisms successively. They
found some strains of bacteria promoted larval growth, although
yeasts did not support its growth. By adopting these assemblages
of microorganisms a high yield was obtained for a prawn larva P. japonicus,
although the success was not always consistent. |
| Douillet and Langdon (1994) have reported use
of probiotics for the culture of larvae of the Pacific oyster (Crassostrea
gigas Thunbeerg). They added probiotic bacteria as a food supplement
to xenic larval cultures of the oyster Crassostrea gigas which
consistently enhanced growth of larvae during different seasons of the year.
Probiotic bacteria were added, at 0.1 million cells/ml, to cultures of algal-fed
larvae, the proportion of larvae that are set to produce spat, and subsequently
the number of spat increased. Manipulation of bacterial population
present in bivalve larval cultures is a potentially useful strategy for
the enhancement of oyster production. They suggest that
the mechanisms of the action of probiotic bacteria are providing essential
nutrients that are not present in the algal diets or improving the oyster's
digestion by supplying digestive enzymes to the larvae or removing metabolic
substances released by bivalves or algae. |
| Maeda and Liao (1994) have reported microbial processes in aquaculture
environment and their importance in increasing crustacean production. They
suggested that based on the photosynthesis of micro algae mainly, it was
clarified that bacteria, protozoa and other microorganisms from microbial
food assemblages use the organic matter produced by the algae and that these
assemblages play a significant role in the aquatic food chain. The growth
of the larvae and their production were markedly promoted by the probiotic
bacteria. In their paper, they also described the presence of a bacterial
clump, stained with a fluorescent dye, inside the digestive organ of the
crab Portunus trituberculatus. |
| Our coworkers of Ecuador considered that use
of different concentration of antibiotics to control the "Zoea syndrome"
of P. vannamei can not obtain a good effect (personal communication).
In their studies using molecular biology techniques, they have concluded
that there exists a relationship between the "Zoea syndrome" and
the presence of bacterial pathogens, V. harveyi, as type E22. The
bacteria strain, Vibrio alginolyticus was found to grow faster
than pathogenic bacteria E22 under all experimental conditions.
For controlling this disease, they used bacterial strain V.
alginolyticus as probiotics in rearing facilities. Their study
shows that the use of probiotics in aquaculture facilities can be an effective
method to prevent disease outbreaks caused by pathogens in shrimp hatcheries. |
| In China, the studies on probiotics in aquaculture
were focused on the photosynthetic bacteria. Qiao Zhenguo et al (1992) have
studied three strains of photosynthetic bacteria used in prawn (P. chinensis)
diet preparation and their effect. Addition of the photosynthetic
bacteria in the food or culture water was found to improve the growth of
the prawn and the quality of the water. Cui Jingjin et al (1997)
have reported on the application of photosynthetic bacteria in the hatchery
rearing of P. chinensis. They used a mixture of several kinds of
photosynthetic bacteria (Rhodomonas sp. ) as water cleaner and
auxiliary food. Their results showed that the water quality of the
pond treated with the bacteria was remarkably improved, the fouling on the
shell of the larvae was reduced, the metamorphosis time of the larvae was
1 day or even earlier, and the production of post-larvae was more than that
of the control. |
| Recently, we have done some research work on
probiotic bacteria in shrimp aquaculture. On the basis of studies on intestinal
micro flora of wild adult shrimp P. chinensis, we have chosen some
probiotic bacteria from shrimp intestinal flora. When the two probiotic
bacterial strains were added to the larval culture water, the survival
rate, the abilities of disease resistence and low salinity tolerance were
improved; average body length and weight were increased. In addition, the
probiotic bacteria, when added to the larval culture water was found not
to influence the total bacterial number and water quality of the sea water.
We also found that some probiotic bacteria can produce some digestive enzymes;
these enzymes may improve the digestion of shrimp larvae, thus enhancing
the ability of stress resistance and health of the larvae (Wang
Xianghong et al 1997, in press). |