Chemical Studies on Freshness of Whale Meat.

IV.

Some Informations of Achromobacter ubiquitum
isolated from Whale Carcass
BY
TADASHI NAKAI(This was reported in Japanese on pp. 152-156, Vol. 71, Journal of
the Pharmaceutical Society of Japan (1951))

Prof. Dr. T. Akiba and his co-operators l) isolated various kinds ofbacteria from sperm whale carcass low in freshness, among which
many strains of Achromobacter ubiquitum were isolated from any ofmuscle, blood and intestines. In order to study what part this bacterium
played in the decrease of freshness in the whale carcass, I took overa strain of this bacterium from him and investigated the decomposing
state of the extract of whale meat by it. Then I searched the acids
produced by the decomposition of glucose by this bacterium. Some
informations obtained are reported hereon.
Experiment and StudyI.

Decomposition of the extract of whale meat

Achromobacter ubiquitum (abbreviated to A. u. bacterium) was inoculated on the extract of whale meat and the decomposing process ofthe extract was traced by determination of volatile basic nitrogenwith the lapse of time.
Preparation of the extract of whale meat :-The refrigerated meatof a whale (Balaenoptera) was minced, to 500 g of which 1 l of physiological salt solution was added and after 3 hours' standing underoccasional stirrings, it was heated in the steam-kettle for 1 hour andfiltered. 1 l of the filtrate was sterilized for 1 hour. (Per 100 cc of
the extract, total nitrogen was 237.4 mg, non-protein nitrogen 226 mg
and volatile basic nitrogen 10.2 mg.)
To the extract obtained in such a way, 0.1 cc of suspension of one
platin loopful of A. u. bacterium for 5 cc of physiological salt solution
was added and incubated at 26""28°C.
Determination of volatile basic nitrogen :-It was determined in
aeration method. To 5 cc of cultural fluid 0.5 cc of saturated K2COa
solution, 0.1 g of NaF and 5 drops of octyl alcohol were added, where to

T. NAKAl

168

air washed through dilute sulfuric acid was sent so violently that thevolatile base was expelled into 0.02 n H2S04. Then the surplus acidwas titrated back with 0.02 n NaOH.
The result is shown in Fig. l. For the first 250 hours, the amount of volatile basic nitrongen (abbreviated to V-N) showed littleii
increase. After that, about till
35
§
"+' l
the 750th hour, it kept the steady
30
:
"q
~
increaseand then decreased sudden8·~ 26
t' ..~
ly. After the decrease from 5.9 to:::: 20
5.5 in pH, increasing again, it was
15 s!l6.5 instantly before V-N showed the1}] 10clear increase. Perhaps the first;1l I •.• .•.
~ 5 o r~o 1100 soo 400 600 600 100 soo 900 iooo decrease in pH is due to the acidbrought from the small amount ofTime in hours
Fig. 1. Decomposition of whale meat ex- saccharide contained in the extract
tract. Figures marked with t mean the by the acid productivity of A. u.
value of pH of the solution at that time.
bacterium. Inspite of no increaseof V-N amount, the value of pH increased in the period about from60th to 200th hour. This probably shows the formation of non-volatileamine. Clearly the generation of gas was recognized after 24 hours
of inoculation. The cultural fluid smelled rather sourish and of its
own, not bad.
0

0

1-o

0

i

II.

Comparison of decomposition between suspension
and extract of whale meat

The state that whale meat was being eroded by A. u. bacterium
was inferred from the decomposing state of whale meat suspension by
this bacterium, and at the same time it was compared with that of
the extract. The whale meat used was of the same kind as I.
Preparation of whale meat suspension :-50 g of whale meat mincedwith the meat-chopper was heated in the steam-kettle for 30 minutes
and brayed in the mortar. To it, 1 l of physiological salt solution wasadded and after 1 hour's heating in tha steam-kettle, its supernatant
was put in another vessel. Its residue was fully brayed again andsterilized for 1 hour after dispersing into the supernatant. Total
nitrogen per 100 cc was 171.1 mg.
Preparation of the extract :-60 g of the above-mentioned whale
meat was heated in the steam-kettle for 30 minutes, and brayed in
the mortar. To it, 1.2 l of physiological salt solution was added. AfterChemical Studies on Freshness of Whale Meat.

IV.

169

2 hours' heating in the steam-kettle and filtering, 1050 cc of the filtrate
was sterilized for 1 hour. Total nitrogen per 100 cc was 36.3 mg.
5 drops of the above A. u. bacterium suspension were added to thus
made suspension and extract respectively, which were kept at 27°C.
The amount of V-N was measured by the aeration method, with 20 ccof each liquid.
The result is shown in Fig. 2. Fluctuation of V-N amount of thesuspension agrees nearly quite with that of the extract. This is, it
is not inf!uertced by the existing
4
amount of muscle protein. This3
fact can be easily understood from ;id
"OOo
""
"o
the known fact that A. u. bacte- .o~rium has no gelatine decomposing S" sbO
power. Therefore, on whale meat ..!!~·~"= 1 tS.q
eroded by A. u. bacterium, nott _.._..._...._..__._...__._....._._..._...._..___.__._
0 ....
muscle protein but mainly nono ioo 200 soo 400 500 600 'i'..OO,
protein matters will be decomposTime in hours
ed. The change of pH in both o-Extract • -Suspension of whale meatliquors agreed about till 300th
Fig. 2·hour (pH 7.1) and after that with an increase of the amount of V-N,
these liquors showed the different value in pH and its difference became
larger and larger. And the then value of pH was smaller in the suspension than in the extract. Probably this is due to protein's buffer
action.
III.

Effect of the initial value of pH upon volatilebase formation

From the above experiments and the general fact that optimum
pH for deaminase of bacteria is alkaline, I imagined this bacterium
deaminated substrate in neutral and alkaline, but scarcely about pH 6.
Therefore, if pH of the extract in the beginning of the experiment
was neutral or alkaline, this bacterium would deaminate the substrate
at once and the amount of V-N would show a clear increase from the
beginning, I imagined. So inoculating A. u. bacterium on the different
extracts in pH, I examined the change of the amount of V-N. The
whale meat used for the experiment was of the same species as iri theabove one.
1.6 l of physiological salt solution was added to 800 g of minced
whale meat and after 3 hours' standing under occasional stirring, boiled

T. NAKAI

170

for 10 minutes and filtered. After ~steam-sterilizing the filtrate for 1
hour, there was inoculated a platin-loopful of A. u. bacterium and kept
standing for one night at 27°C. Thus, fermentable matter (carbohydrate) in the extract was consumed, to preventing as much as possible
from decreasing pH instantly after the commencement of the experiment as in experiment I and II. And through the layer of kieselguhr,
bacteria were sucked off. Each 480 cc of the filtrate was made at 6,
7 and 8 in pH and after 30 minutes' heating in the steam-kettle,changed pH was adjusted with NaOH and added physiological salt
solution made its amount 500 cc. It was put in a 1 l Erlenmeyer's
flask and sterilized for 30 minutes every day for the following three
21
~

20

]
z·= 15

0

100

200
400

'Time in hours
Initial value of pH in the extract :x-6, o-7, . • -8
Fig. 3.

600

Time in hours

Initial vulue of pH in the extract :x-6.1, a-6.9 • -7.9
Fig. 4.

days. To each of them, 0.1 cc of the above
mentioned• suspension of bacteria wasadded and kept at 27°,......,28°C. Often lOcc
of this solution was taken out asepticallyto measure the amount of V-N.
The experiment was carried out twice
and its result is shown in Figs. 3 and 4.
In the extracts of 7, 6.9 and 8, 7.9 in pH
at the beginning of the experiment, theamount of V-N increased from the outset
but in the extracts of 6, 6.1 in pH it did
hot increase in the early stage of theexperiment with even a decrease until
towards 7 in pH. This result seemed tocoincide fully to my expectation before
the experiment. But in case of peptone

35

25

"'

·~
..0

15

v

5 f.____._ __,__ __,__ _,
0

100

200

Time in hours
Decomposition of pepton salt
solution. Intial pH :x-4.9
o-7.1 • -8.1
Fig. 5.

Chemical Studies on Freshness of Whale Meat.

IV.

171

solution (1% of Polypepton "Takeda", 0.5% of NaCl) used as cultural
medium, in any of 5.9, 7.1 and 8.1 in the initial pH, the amountof V-N increased from the outset and the increase of the amount of
V-N at 5.9 in pH was the most till 162nd hour (Fig. 5). Therefore,
we can consider that peptone contains some substance that can givethe volatile base by A. u. bacterium's action even at 6 in pH but
the extract of whale meat does not contain it.
In the extract of whale meat, at a certain stage the amount of
V-N decreased in sudden with state of equilibrium in conclusion. In thisstate the amount of V-N was nearly same, with no relation to pH in
the beginning of the experiment (Fig. 4).
IV.

Decomposition of autolyzate of whale meat

In the decreasing process of freshness of whale carcass, prior to
or at the same time with the decomposition by bacteria, autolysis ofmuscle takes place and intermediary products of protein decomposed
increase gradually and the amount of the extractive matter also. To
find out the relation between it and the A. u. bacterium's action, theextracts of autolyzed and normal whale meat were made and theirdecomposing state by A. u. bacterium was compared.
The preparation of the extract of normal whale meat (control solution) :-To 500 g of minced sperm whale meat, 1 l of physiological salt
solution was added and after 3 hours' standing, it was boiled for 10
minutes. The filtrate was sterilized for 1 hour and a platin-loopful of
A. u. bacterium was inoculated on it and kept at 28°C for a night.
Thus, after consuming the acid-fermentable matters, the solution was
sucked through the kieselguhr layer and the filtrate was neutralizedwith NaOH, of which 500 cc was put in a 1 l Erlenmeyer's flask andsterilized for 30 minutes every day for the following 3 days. Total
nitrogen was 117 mg per 100 cc.
Preparation of the solution of autolyzate :-To 500 g of mincedmeat of sperm whale, 1 l of physiological salt solution saturated withchloroform and 5 g of chloroform were added and kept at 27° ,_28°C
for 5 days. Then, after 10 minutes' qoiling, the filtrate was treatedas mentioned above. The total nitrogen was 190.3 mg per 100 cc.
0.1 cc of A. u. bacterium suspension was added to thus obtained
two kinds of solutions and kept at 27°,_28°C. The result was shownin Fig. 6.
The amount of V-N was always more in the solution of autolyzate
than in the control solution but both solutions showed nearly same

T. NAKAI

172

changing state till about 200thhour. However, after the 200th• '°25.
hour, the control solution showed28
little increase of the amount of
V-N and then gradual decrease,but the solution of autolyzate
showed the remarkable increaseagain. Can't we interprete thisphenomenon as follows ? In thesolution of autolyzate, kinds ofsubstances decomposed by A. u.
bacterium were different between
500
400
300
100
200
till
and after about the 200th
Time in .hours
Fig. 6. Comparison of the decomposing pro- hour ("The substance decomposedcess of the extract between autolyzed by A. u. bacterium" is limited towhale meat and normal whale meat.
the substance forming the volatile• , o-Autolyzed, a-Normalbase by the action of A. u. bactetrium, in this case). Till about 200th hour, the substance decomposed
by A. u. bacterium was of same kind in both solutions and consumed
almost all by this time. After this time, the control solution had no
substance decomposed by A. u. bacterium, so the amount of V-N did
not increase. In the solution of autolyzate, A. u. bacterium foundanother kind of substance to be newly utilized in the product of autolysis and began to decompose actively and the amount of V-N increased
again.
As in the preceding experiments, both solutions showed the sudden
decrease of the amount of V-N in a certain stage.
27,4,qaea"'n"'of(1%/31S
011 bottom

0

V.

•

0

Effect of A. u. bacterium upon the decreasing
freshness of whale carcass

The results of the above experiments can make us imagine as follows :-No A. u. bacterium decomposes protein and gives out the badsmell. So if this bacterium only acts on the whale carcass, it gives
no remarkable effect on freshness. However, generally speaking, inthe decreasing process of freshness, only one species of bacterium acts
rarely, and simultaneously various kinds of bacteria work. So in sucha case, A. u. bacterium decomposes actively various intermediary decomposition products of protein which are produced by other bacteria,
especially bacteria with protein decomposing power, and gives abundantly the last products of protein decomposed. Namely it can be safely

Chemical Studies on Freshness of Whale Meat.

IV.

173

said that A. u. bacterium can not become the main cause of decreasing
freshness on whale carcass but plays a part by participating in thelast process of protein decomposing.
VI.

The cause why the sudden decrease in the amount of V-N takes
place in a certain stage of decomposing process of extract

In most of the above experiments, the sudden decrease of theamount of V-N was found in a certain stage. The pH was always
bigger than 7.5. From the further shape of the curve we can judge
that this decrease is not caused by volatilization. After my incessant
observation, I found that in this50
stage fine sand-like or short prismal crystals appeared on the bot- g
tom of the vessel without fail and s 40
Appear.;mce ot
they increased their number with ~cr_ystals.sthe decrease of the amount of z
30
V.,..N (Fig. 6 and 7). Wondering if~
this crystal had any relation withthe decrease of the amount of
V-N, I investigated to find that
this crystal contained ammonia as
my expectation.
10
100
0
400
200
80Q
This crystal is an inorganic
Time in hQur$
soluble insubstance, hardly
Fig. 7. Appearance of crystal ofwater and alkali carbonate soluNH,MgPO. · 6H20 and decrease oftion and readily in dilute mineral
the amount of vatile basic N
acids. Its dilute mineral acid solution, if it is alkalified with NaOH,
gives the gelatinous precipitate and generates ammonia. This crystal
was conffrmed to be magnesium ammonium phosphate from the factof containing Mg·· and P04"' as well as ammonia and the content ofphosphorus. 'rhe determination of phosphorus was done by Bell-Doisy's 2)colorimetry with solution of the crystal in 0.01 n HCl as test solution.
Sample :1.63 mg,1.51 mgp :o.204 mg, 0.188 mg
NH4MgPQ4 · 6H2 0 : calculated 12.63
found
12.52,
12.45
Accordingly, the cause for decrease of the amount of V-N was
found on the crystallization of magnesium ammonium phosphate. It
was certificated by its crystallization even in the extracts in Fig. 1
and Fig. 7 which were made without the treatment through kieselguhr.
000T. NAKA1

174

VII.

Acids as the metabolic products of Glucose by A. u. bacterium

A. u. bacterium produces acids from saccharide. Acids produced
aerobically from Glucose were searched.
Preparation of cultural medium :-The solution of 1 l of whale broth,
10 g of Glucose and 5 g of NaCl were neutralized with NaOH and aftrsterilization, added 5 g of CaC03 separately sterilized. A 2 l Erlenmeyer
flask was used as vessel.
A platin loopful of A. u. bacterium was inoculated on this cultural
medium and incubated at 28°,.,.,,30°C for 3 days under occasional stirrings.
2 l of thus fermented solution was treated as the following table anddivided into the volatile acid solution, ether-soluble non-volatile acidsolution and ether-insolubie acid solution.
2 l of fermented solution
I

I

I

Precipitate

Filtrate

decomposed withdilute H2S04

I

Filtrate+-~extracted
with ether

add

~

I
I

Etheric soluion

Ether distilled

slightly alkalized, concentrated into· about 150 cc onwater-bath, decompsed withdilute H2S04 and centri. fuged.

~

I

Supernatant
I

Residual solution(Ether.insoluble acid solution)

I

Residue

Steam-distillation
I,-----------------[

Distillate(Volatile acid solution)

Residual solution(Non-volatile acid solution)

I tried to isolate acids from the above each solution.
Volatile acid :-To volatile acide solution, PbO was mixed in excess
ond evaporated to dryness on the water bath and the residue was infused with about 35 cc of tepid water. The filtrate was heated in theviolently boiling water-bath but no basic lead propionate was found.
Therefore, after removing lead by adding dilute H2S04 to the filtrate,
ZnO was added and evaporated to dryness on the water-bath. The
residue was infused with absolute alcohol after two hours' desiccation
at 150°C. To the residue, phophoric acid was added and distilled withsteam. The distillate showed negative reaction against formic acid.
Alcohol distilled off the infusion and . after adding phosphoric acid toChemical Studies on Freshness of Whale Meat.

IV.

175

the residual solution, it was distilled with steam. Though white substance was found in the distillate, its scarcity made the further investigation impossible. Then it was filtered off. The filtrate wasneutralized with NaOH and concentrated into about 3 cc on the waterbath and added saturated AgN03 solution. Instantly after, whitesubstance appeared, which was filtered out and recrystallized fromwarm water. About 0.2 g of needle crystal of silver acetate wasobtained.
Ag 43.8mg
Sample 68. 7 mg
CH3C00Ag
calculated 64.64
found 63.75
Non.volatile acid :-Non-volatile acid solution was neutralized withsaturated Ba(OH) 2 solution and concentrated on the water bath. 25 grof the concentrated viscous substance was dissolved into 250 cc of 80%
alcohol under warming and after cooling, it was filtered. The residue
was inorganic. After distilling alcohol off, the filtrate was diluted
with water and decomposed with dilute H2S04 and filtered. The filtrate
was extracted with ether and then it was distilled off. After thevolatile substance which somewhat seemed to be contained in the residue
was expelled off through steam-distillation, some water and ZnC03 in
excess were added and heated on the water-bath and filtered. The
filtrate was treated with active carbon and concentrated on the waterbath. About 10 g of crude zinc lactate was obtained. After recrystallizing from hot water and desiccating at the room temperature, theyield was 5.4 g. Some more crystals were yielded from the motherliquor.
Sample 298.2 mg; 215.5 mg (yielded from the mother liquor)
Loss on heating at 115°C 53. 7 mg. 39.0 mg
ZnO
81.7mg; 58.7 mg
Zn(C3H50 3k 3H20
calculated crystal water
18.16
ZnO
27.35
found crystal water 18.01 ; 18.1
ZnO
27 .43 ; 27 .24
According to this analysis value, the lactic acid produced by A. u.
bacterium is racemic mixture. Therefore, A. u. Bacterium is imagined
to contain racemiase. 3J
Ether.insoluble acid :-It was searched with newly made fermented
solution. It was concentrated on the water-bath and decomposed withdilute H2S04 and centrifuged. The supenatant was extracted withether for removing the ether-soluble acids and the residual solution
was boiled with ecx.:cess CaC03. In the filtrate and the residue, acid176

T. NAKAI

was searched but no organic acid could be found.
Accordingly, organic acids found as metabolic products qf glucose
were considerable amount of lactic acid and minute amount of aceticacid.
In conclusion, sincere thanks are expressed to Dr. T. Maruyama
for his kind advice and to Prof. Dr. T. Akiba for transfering the precious bacterium and to Mr. H. Okuda for revision.
Literatures
1) Sci. Rep. Whales Research Institute No. 3, 63 (1950)
2) J. Biol. Chem. 44, 55 (1920)
3) H. Katagiri, K. Kitahara·: Bull. Agr. Chem. Soc. Jap. 12, 127 (1936)