t1
e ARCHIVES FISHERIES AND MARINE SERVICE
Translation Series No. 3723
Microbiology and biochemistry of brines and microbiology of cooking hams
by C. Cantoni, M.A. Bianchi, S. D'Aubert, and P. Renon
Original title: Microbiologia e biochimica delle salamoie e microbiologia dei prosciutti da cuocere.
From: Arch. Vet. Ital. 19(6): 401-423, 1968.
Translated by the Translation Bureau( MMM ) Multilingual Services Division 1'. Department of the Secretary of State of Canada
Department of the Environment Fisheries and Marine Service Biological Station St. John's, Nfld.
1976
34 pages typescript (
, DEPARTMENT OF THE SECRETARY OF STATE SECRÉTARIAT D'ÉTAT TRANSLATION BUREAU BUREAU DES TRADUCTIONS
MULTILINGUAL SERVICES eiee.;;'t DIVISION DES SERVICES CANADA DIVISION MULTILINGUES
• r n7 1/-3'7D _p TRANSLATED FROM - TRADUCTION DE INTO EN Italian English
AUTHOR -• AUTEUR Carlo CANTONI et al.
TITLE IN ENGLISH - TITRE ANGLAIS Microbiology and Biochemistry of Brines and Microbiology of Cooking Hams
TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTERES ROMAINS) Microbiologia e Biochimica delle Salamoie e Microbiologia dei Prosciutti da cuocere.
REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. RÉFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÉRES ROMAINS. Archivio Veterinario Italiano
REFERENCE IN ENGLISH •••• RÉFÉRENCE EN ANGLAIS Italian Veterinary Archive
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BUREAU NO. LANGUAGE TRANSLATOR (INITIALS) N° DU BUREAU LANGUE TRADUCTEUR (INITIALES)
May 25, 1976 1101124 Italian
Institute for the Inspection of Foodstuffs of Animal Origin of the University of Milan, Italy (Director: Prof. Giovanni Giolitti) UNEDITED TRANSLATION - • For Wormaflen on!--/ MICROBIOLOGY AND BIOCHEMISTRY OF BRINES TRADUCTION NON REVISEEE-: • AND MICROBIOLOGY OF COOKING HAMS Information souiomont BY Carlo Cantoni; Maria Antonietta Bianchi; Simona D'Aubert & Pietro Renon
Summary - Reported hereunder are the analytical data regarding the chemical
and bacteriological composition of brines for cooking hams and the
bacteriology of cooking hams.
Halotolerant germs and isolated halophiles are described. Their
importance in salting is discussed and described.
The preservation of salted meats by means of salting is a technique
of extremely ancient origin : substantially there are two methods which
are commonly used to-day: dry salting and immersion in brine.
The dry salting techniques are mainly used for purposes of lengthy
aging and preservation; usually it is preferable to use the brine technique
for products which undergo further processing stages (for example brine
techniques are used for products like bacon and brisket).
os-zoo-I 0-31
• I% I • 11. . •,1 Dry salting is effected either by lengthily rubbing salt over the
entire surface, of the meat so that it is absorbed, or by alternating
layers of meat and salt in vats or tubs. In this case, the liquids
extracted from the meat through the effect of osmosis in time, form, a real
brine in which the meat is partially immersed. In cooking ham, salting is
generally effected by injecting the brine into the blood vessels or muscles
of the ham, which is then placed in vats containing the saline solution.
For a more detailed description of the methods in use, we would refer the ready to Ghinelli (1951).
During recent years in various countries, much research work has
been carried out with the purpose of pinpointing the biochemical and
biological phenomena which occur during the salting of meats of various
animal species (cattle, hogs and fish): already in 1967 a symposium was
devoted to this problem and its proceedings contained information of
considerable interest. In addition, various researchers, among whom we
shall mention Leistner, 1959; Ten Cate, 1962 a, b, c, d; 1963 a, b;
Molinari, 1964; Patton & Wilson, 1965; Taylor & Walters, 1967; Barbe &
Henricson, 1967; have contributed further to the knowledge of this subject.
In our own institute, extensive research has been carried out on sausages (Cantoni, 1964 a, b; Massacra, 1964; Cantoni & Massacra, 1964;
Cantoni et al., 1967) so that at the present time, we have quite extensive information available in this field. •.
It is considered that the preservative action of salt, normally
constituted by sodium chloride with the addition of nitrates (and sometimes
of nitrites) applied with the dry method or with brine, consists of
removing water from muscular tissues through high osmotic pressure,
diminishing the levels of the free water (thus preventing the multiplication
of non-halophile or halotolerant species and depressing the activity of the
enzymes), and forming red nitrosopigments (nitromyoglobin and nitrosohemo-
globin) through the action of products of microbial or enzymatic destruction
of the nitrates (or nitrites) added to the salt (Walters & Taylor, 1968).
While the formation of nitrosopigments in the cooking hams has already been
clarified exhaustively from the biochemical viewpoint by the two authors
cited, it does not seem that there are concordant opinions on the microbial
species responsible for the reduction of nitrates to nitrites, so that it
is still not clear if this process should be attributed to one or more
microbial genera amongst those present in the brines, nor has it been
possible entirely to evaluate their importance.
According to French authors (Buttiaux, 1957; Henry, 1957) the
flora active in the reduction of brine nitrates consists mainly of a single
microbial genus: that of the halophile vibrions.
In Wiltshire bacon brines, the most numerous germs present on the
surface are the micrococci (Garrard & Lockhead, 1939; Ingram, 1952), while
other microorganisms, like the clostridia, the streptococci, the entero-
bacteria and the Alcaligenes, are present in lesser number. Again with
regard to bacon brines, more recently Ingram (1962) observed that the
predominant genus is the Achromobacter spp., and he furthermore considers
that other halophiles, difficult to isolate, may play a primary role. • f 4
United States researchers, on the other hand, do not consider
microbial action very important, but, they do howevèr, observe that the
germs present in greater number in brines include ai least 23 microbial
genera: the micrococci, the vibrions, the Achromobacter, Brines used in
the United States have an exclusively nitrite base:
Leistner (1959) states that in the brines of hams produced in
Germany at least 23 microbial genera are present: micrococci, vibrions,
the Achromobacter, streptococci and the Alcaligenes are the most numerous.
The Irish researchers Patton & Wilson (1964) indicate the presence
of a great number of gram positive elongate halophile germs.
Ten Cate (1962) states that the micrococci are the predominant
flora in Dutch brines.
The Polish researchers Wichlacz & Wislouch (1968) after indicating
the predominance of halophile vibrions, of Flavobacterium and Achromobacter,
not that when the brines of cooking hams are inoculated with cultures of
Vibrio spp., the color of the meat becomes more intense, whilst the
addition of cultures of Achromobacter and Flavobacterium improves the
organoleptic qualities of the ham.
Recently Gardner & Patton (1968), in a further investigation on the
microbial flora of bacon, state that the predominant flora consists of
Micrococcus, Acinetobacter and Vibro spp.
While in various countries the problem has been studied and
thoroughly investigated, only sporadic works have been published by us on
this subject and we therefore thought it worthwhile to start a series of
research works on the salting process of cooking hams produced in our
country (Italy). Some preliminary'results have recently been published (D'Aubert et al., 1968; Cantoni et al., 1968).
In this paper, we intend to report the data on an extensive microbiological and chemical experiment conducted simultaneously on brines and on cooking hams.
Materials and Methods
The following chemical analYses were carried out on 48 samples of different brines, weighing respectively 5 gg (5 samples), 10 gg (26 samples),
15 gg (7 samples), 26 gg (10 samples):
pH (by means of the Ionosis meter model pH Q 4 pH); total nitrogen: by the microKjeldhal (method) with mineralization according to Beet (1954); amino nitrogen: by the Michel method (1960); free and fixed ammonia: by
the Conway method modified by Engst (1964); lactic acid by the following method: added to 10 ml of brine are 10 ml of CuSO4 at 20% and Ca (OH2) in powder, which is there let stand overnight, centrifuged, the supernatant removed and the lactic acid determined with concentrated sulfuric acid and hydroquinone according to Snell & Snell (1965); sodium nitrite: by the
Benassi method (1966) sodium chloride: by the Volhard method (Jacobs, 1958).
Microbiological tests were conducted on 45 brines, on 26 of them 8 days, on 7 of them 15 days and on 10 of them 20 days, from the time of use.
For the numbering of the microorganisms, decimal dilutions were carried out in a solution of tryptone salt containing 1 g of tryptone and
75 g of salt per hundred ml of distilled water (1) ;0.1 ml of every dilution was placed on the plates of the various mediums and spatulated over the 0 entire surface. The cultures were incubated at 30 (c.) for five days.
(1) This concentration of salt was used bearing in mind that in the brines the concentration in NaCL is high and that consequently modifications also to the composition of the microflora would not have occurred. . 6
The total bacterial count was effected on tryptose agar; the germs of the genus Achromobacter-Alcaligenes were isolated:on agar-saccharose-salt
(Buttiaux, 1957) and tryptose agar containing 1 U/ml of sodium penicillin, the Enterobacteriaceae on agar deoxycolate-lactose (Leifson, 1935) the
Micrococcaceae on mannitol-salt agar (Chapman, 1946) the Lactobacillus on
RMW medium (Rogosa, Mitchell & Wisemann, 1951) the D streptococci on Barnes medium (1956), the Bacillus on tryptose agar, the Clostridium on VF-sulfite medium (Buttiaux, Beerens & Tacquet; 1963). For the halophile microorganisms,
Buttiaux medium was used (1967), as well as a medium made up as follows:
Difco no. 3 proteose peptone g 5, Oxoid yeast extract g 10, Difco Casamino- acids g 5, KC1 g 2, MgSO4 g 10, KNO2 g 1.8, NaC1 g 60, Oxoid Agar g 15, gelatine g 4, distilled water ml 1000, pH 6.7, sterilization 1210 (C.) for
15'.
For the study of the microflora of the hams, samples salted in different brines were used. Removed from each of these, after sterilization of the external surface, were portions of deep muscle of approximately 20 g which were homogenized with a solution of tryptone salt (tryptone 0.1%,
NaC1 7.5%) in the ratio 1:4 (p/v); and subsequently we proceeded in the same way as for the brines.
For the maintenance of the isolated strains (the halophilic or halotolerant characteristics had previously been verified on the same maintenance substrates, but containing decreasing quantities of NaC1) the following cultural mediums were used: Micrococcaceae: common agar + 2%
NaCl; Streptococci D: tryptose agar; Lactobacillus: M.R.S. mçdium (De
Man, Rogosa & Sharpe, 1960), and Robertson's medium (Mackie & McCartney,
1965). Bacillus: nutritive agar; Pseudomonas: King B medium (King et al., 7
1954); Corynebacterium: medium described in Cantoni et al. (1957);
Achromobacter-Alcaligenes: nutrient agar; Vibrio: saccharose-salt agar
(Buttiaux, 1957); Halophiles: the medium indicated above was used but without the KNO2.
For the identification of the isolated strains the following methods were adopted:
Staining: Gram according to Buttiaux et al., 1963, Rhodes' staining (cited in Buttiaux et al., 1963, staining of the spores according to Shaeffer & Fulton (1933); staining for the halophiles according to
Cantoni et al., (1966).
Micrococcaceae: the schema proposed by Baird-Parker (Gibbs &
Skinner, 196... (sic)) was followed.
Lactobacillaceàe: a) Streptococcaceae: Sherman's criteria as reported by Buttiaux et al., 1963, were followed; b) Lactobacillus:
Sharpe's method (1961) was followed.
Corynebacterium: the schemas and methods indicated by Breed, Murray
& Smith (1957) were followed.
Bacillus: the methods of Smith reported by Cowan & Steel (1965) were adopted.
Clostridium: only the presence of Cl perfringens was verified, using the methods reported by Mossel, Bechet & Lambion (1962).
Pseudomonas: the methods reported in Buttiaux et al., (1963) were followed.
Halophiles: the methods of Penso et al. (1967) and those reported by Monti & Cantoni (1968) were adopted. I 8
Vibrio: the methods reported by D'Aubert et al. (1968) were
followed. All the isolated strains, halotolerant and halophile, were tested
for their capacity to reduce nitrates to nitrates and the latter to N2 , NO,
NO2. For this purpose, the cultures were placed in test tubes of broth-
nitrate at 6% NaCl, with a Durham bell and incubated at 30°C for 5 days.
During, and at the end of, this period, we evaluated the production of gases,
and for the negative strains, also the reduction of nitrates to nitrites in
accordance with Cowan & Steel.
The denitrifying strains were present in the halophile group: we
proceeded to their classification and to the study of this characteristic
to determine whether it depended on a respiratory or on an assimilative
phenomenon, and to evaluate its effect on the success of the salting.
For the study of denitrifying activity, the two most active strains
belonging to the percentually most representative groups were selected.
The following mediums were used:
1) Medium A: mono-ammonium phosphate gr. 3; K2HPO4 g 1; glucose g
3; NaCl g 50; KNO3 g 2; KCL 0.5; MnSO4 traces; MgSO4 g 0.2; FeC1 traces;
distilled water ml 1000; pH 6.9; sterilization 20' at 120°C.
2) Medium B: same composition as the preceding one with 2% of yeast
extract, sterilization for 20 minutes at 120°C.
3) Medium C: same formula as the p'receding one with 14% NaCl.
Sterilization for 20 minutes at 120°C.
The three mediums distributed in flat-bottomed conical vacuum flasks
closed with a ground (frosted) glass stopper, were inoculated with the two
strains under examination and left to incubate for 120 hours at 5°C and at
30°C. 9
Subsequently the same series of tests was repeated with the same mediums after a depression equal to 360 mm of Hg had been created. The tests listed hereunder were carried out at intervals of 24, 48, 72, 96,
120 hours.
Gas chromatographic determination of the gas products: (CO2; 02;
N2+N0+NO2). For the analysis, an amount of gas was removed through an air- tight membrane applied to the lateral tube of the vacuum flask with a
Hamilton gas syringe: on one side the 02 and the sum of N2 + NO + NO2 were metered and the CO2 on the other.
The gas chromatographic parameters used were the following:
1) For the determination of 02 and the sum of N2 + NO + NO2: 5 o Angstrom sieve-plate column; length of column 2 meters; operating 2 temperature of column 20oC; pressure of carrier gas (hydrogen): 0.26 kg/m ; quantity of gas introduced: 4-5 ml; velocity of paper 10 mm/minute; heat conductivity detector (filaments).
2) For the determination of the CO2 and the sum of 02 + N2 + NO + NO2:
Chromosorb 102 column; length of column 2 meters; operating temperature of column 20°C; carrier gas pressure (hydrogen) 0.23 kg/m2 ; quantity introduced:
4-5 ml; velocity of paper: 10 mm/minute; heat conductivity detector
(filaments).
Determination of the nitrites formed in the broth cultures: the method reported by Benassi (1966) was used. 10 •
Evaluation of microbial growth: the microbial growth was evaluated on the basis of the total nitrogen content of the microbial cells, metered with the microKjedhal through mineralization according to Beet (1954), separated through centrifugation of the culture medium and washed three times with physiological solution.
Determination of the free ammonia: the ammonia present in the broth culture was evaluated with the Conway method modified by Engst (1964).
Determination of the pH: this was effected as already described.
Finally in order to evaluate the importance of the effect of denitrifying strains of microbes on the concentration of nitrites in the brines, strains belonging to different groups of denitrifiers in the two following mediums were examined:
Medium D: the smne as medium A, but with 14% of NaCl;
Medium B: the same as the maintenance medium of the halophile germs, but with 14% of NaCl.
The broth cultures were left to incubate at 5-8°C, under vacuum, for 30 days. The production of gas was controlled visually on a daily basis; at the end of the test the sodium nitrite was metered.
Results
In tables 1-7-8-9, the results of the chemical investigations are shown: the average concentration of sodium chloride was 14.2%; 16.3%; 21.1%;
14.3%; in the brines 5, 10, 15, and 30 days old, respectively. 11
The pH in all the samples examined did not show wide variations in
the average values, keeping, in fact, close to neutral (values of 7-7.4).
The nitrites have almost the same value in the brines 5, 10, and 15 days
old; they decrease to values of 4 mg/1 in those 30 days old. The total
nitrogen content gave values between 73.9-164 mg/100 ml. The highest values
were found on the 5th and 30th day.
The amino nitrogen displayed values of between 3.2 and 30.9 mg/100
ml. These values increased from the-5th to the 15th day, and subsequently
diminished.
The values of the free and fixed ammonia increased constantly, the
free ammonia varies between 1 mg/100 ml (5th day) and 1.6/100 ml (30th day),
the fixed between 64.5 and 223.3 mg/100 ml.
The concentration of lactic acid increases constantly from average values of 100 mg per 100 ml in 5 day old brines to 321.6 mg per 100 ml in
those 30 days old.
The results of the microbiological investingations are summarized in
tables no. 2, 3, 4, 5 & 6.
The total average number of halotolerant germs was 1,769,412 per ml
in the 5-day brines, 6,650,000 in 15 day-brines, 11,048,570 in 30-day brines,
and thus we note a progressive increase in their number with the age of the
• brine.
They appear to belong to the families: Micrococcaceae (Sarcina and
Micrococcus) , Corynebacteriaceae (Corynebacterium) , Lactobacillaceae
(Lactobacillus and Streptococcus D, according to Lancefield), Pseudomonadaceae
(Pseudomonas) , Achromobacteriaceae (Achromobacter-alcaligenes) , Entero-
bacteriaceae (the classification within this family was not gone into very 12
deeply, but we feel that the strains isolated must be considered conform),
Bacillaceae (Bacillus).
The halophile microorganisms capable of developing on culture mediums are less numerous than the halotolerant ones: 1,130,000 per ml in the 5-day brines 3,160,000 in 15 day-brines, 3,680,000 in 30-day brines.
They belong to the families of the Micrococcaceae (Micrococcus and Sarcina),
Spirillaceae (Vibrio), Pseudomonadaceae (Halobacterium). The halophile microorganisms may be roughly divided-into two groups: the first includes the round or rod-shaped ones which reduce nitrates to nitrites and no further, the second the round or rod-shaped microorganisms, which reduce the nitrates to the end products: N2, NO, NO2 .
Falling into the first group are the sarcinae, the vibrions (strains were isolated belonging to groups I-II-III-IV-V of the XI described by
D'Aubert et al., 1968, Cantoni et al., 1967, Bianchi et al., 1968) and 100 tribes temporarily grouped under the denomination Halobacterium (rod-shaped) and Micrococcus (round in shape).
Table 1
Average values of pH, total nitrogen, amino nitrogen, free & fixed ammonia,
lactic acid, concentration of NaC1 and nitrites, in the brines in cooking hams
pH Total Amino Free Fixed Lactic NaC1 Nitrite nitro- nitrogen Ammo nia Ammonia Acid gen mg /100 mg/100 mg/100 ml mg/100 ml mg/100 mg/100 mg /100 ml ml ml ml ml
5 5 5 5 5 5 cll X cd (Si a "1 ind :1 E m 0 0 a.) e bi) to .23 bO ozs oes he e b0 ozs b0 bo cd H cd cd cd cd cd cd cd E cd S-1 P 5 o 0 › 0) › of t 0 x › E ‹ .ri
0 ‹ •ml
e 0 0 0 Ag
8.1-22.3 4.1 14 -10.5 5 gg 7.13 7 -7,3 1313 70-238 3,23 1.5- 5 1.6 0,72-3,50 223,3 31 -331 321.6 83-620 14.3
7.4-25.9 11.6 4.3-16.8 10 gg 6 7 6.2-8,3 89,2 11-473 4,1 0,2- 28,5 0,05-1,78 64,5 23,5-180 100 70-140 14.2
15 gg 7,4 6,3-8,3 73.9 12-163 30.9 0.7-177 1,3 0,02-3,6 69,3 0.5-324 114.2 52-676 16.3 8.3-23.1 12.4 4,1-15.6
30 gg 10 7,3 6,3-8,5 164 63-3M 9,6 3,1- 16.1 1,7 0,72-2,16 123.7 27 -243 259,4 110-392 21.1 16,2-26.3 12 3 5,1- 5,3. Table 2
Average microbial counts of brines of cooking hams of different ages
No. of brines examined 26 7 10
5days 15 days 30 days age of the bine
average max. & average max. & average max. & no. of min. no. no. of min. no. no. of min. no. colonies of colonies colonies of colonies colonies of colonies
Total count 17x 10 3 x 10'- 3 x 10' 66x 10' 10 x 1042 x 10' 110 x 10' 10 x 10'40 x 10° Micrococci ' 91 x 10' 8 x 10'- 3 x 10' 26 x 10' 12 x 1W- 4 x 10° 86 x 10' 19 x 180-28 x 10' Enterobacteriaceae , 11 x 10' 10 -78 x 103 10 x 10' 100- 5 x 10' 25 x 10' 2 x 10% -3 x 10' - --. --.. Halophiles (nitrite medium) . 11 x 103 22 x 10'41 x 10' " 31 x 10' 25 x 103- 3 x 10' . 36 x 10' 23 x 103- 9 x 10' ' Halophiles (Battiaux's medium 12 x 10' 25x10- 3 x 10' 30 x 10' 31 x 10'- 5 x 10' 32 x 103 17 x 10 3- 9 x 10' • Streptococci D 12 x 10' 100 -25x 10' 18 x 103 100- 9 x 10' 28 x 102 100 -13 x 10 2 Lactobacilli 93 x 10' 100 -50 x 10' 43 x 10' 3 x 105-20x 10' 41 x 10' 1000 -13 x 10' ' Sulfite reducing clostridia 3 0-5 8 0-18 4 0-6 • '. 15
• Table 3
Average microbial contents of raw cooking hams (germs/gram) - Samples taken from deep muscles. Number of samples examined: 9
Average no. Maximum & Minimum of colonies no. of colonies
Total count , 60 x 10' 15 x 10 - 14 x 10' Denitrifiers 26x 10' 75 x 10' - 55 x 10' Micrococcaceae 52 x 10' 5 x 10' - 15 x 10' Halàphiles (Buttiaux's medium) 11 x 10' 15 10' - 30 x 10' Enterobacteriaceae - 5 x 10' 0 - 1fr . Streptococci D 33 x 10' 1F' t ' 10' - 10 x 10' . Lactobacilli 35 x 10, 45 10' - 10 x 10'
In the second group (which comprises the germs able to reduce nitrates to N2 , NO, NO2 ) over and above the vibrions, it was possible to distinguish, six subgroups on the basis of their morphological appearance and some biochemical characteristics (table no. 4).
The halophile cocci (Micrococcus and Acinetobacter) are round microorganisms, drranged individually or coupled or in irregular clusters.
Their size varies from strain to strain and even in a single strain depending on the age of the culture and the conditions of growth. They are immobile, asporogenous, heterotrophic, aerobic, they grow in mediums with NaC1 concentrations of 2-15%, at a temperature beiween 8 °C and 30°C.
They have in common the properties of not hydrolyzing urea, of not decarboxylating lysine, of not producing indole and H2S (*)
(*) According to Bergey's Manual (1957) the halophile cocci are classified partly under the genus Micrococcus, partly under the genus Sarcinae; recently Bohacek, Kocur & Martinec (1968), on the grounds of the study of the basic composition (G/C) of thebNA in these microorganisms proposed including all the halophile micrococci in a new genus: Halococcus; in this work we are still using the terminology of Bergey's Manual. 16
The behavior of the isolated strains both as regards the appearance of the colonies, and as regards some biochemical characteristics, has enabled us to combine them into four subgroups (see table no. 5)..'
The rod-shaped halophile microorganisms have the following character- istics: Gram microorganisms -, rod-shaped, showing a certain degree of pleomorphism. They do not form spores, they are mobile and polarly flagellate. They grow optimally in mediums with a NaC1 concentration of
2-15%. They grow optimally at temperhtures of 8°C-30°C. They are hetero- trophic, optionally aerobic-anaerobic, they can grow in the presence of ammonium salts only. (In Bergey's Manual of Determinative Bacteriology similar microorganisms, characterized by a greater halophilia, are included in the genus Halobacterium).
All the strains isolated reduce nitrates to N2-1-N04-NO2. They do flot
do not decarboxylate lysine and they do not produce produce indole, they
H2 S. On the basis of biochemical and morphological tests, it was possible to distinguish two subgroups: 17
Table 4
Classification of the microbial strains isolated from the brine of raw cooking hams and their capacity to reduce nitrates to nitrites and to N2 + NO + NO2
Number and percentage of strains which reduce nitrate to NITRITE and N2 + NO2 + NO
4J 'ri — c.) i Sarcina spp. 15(MO%) .0 0 Gram /)1icrowecus spp. 15(100%) 0 positive l Streptococeas spp. 2(M0%) 0 W• w Lactobacillus spp. 10( 0%) 0 P W 0 4..) srp. 8( 0%) 0 spp. 5(100%) 0 m cJ 0 Pseutloinon ■ s spp. 10(100%) 0 T-1/-4 qj W Gram Achrontobarter spp. = bl)e Alcaligenes 110 ( 65%) 0 negativel Coliforms 5(100%) 0
• Cf] Sarcina $pp. group. I 20(100%) o El 0 . 0 • /-1 roup II 15(100%) W 4 rOUp III 7(100%) o group IV 14(100%) 0 0 w Vibrio group V m (1o%) o 1 spp. (1 r-1 •r-1 alobacterium18 00%) 0 i 4 1-1 icrococcus . .• P.■ C.) er-I and Unident4000 o !,, O 0 0 P-1 .-cd . if ied cd 0 0 ..i Micrococcns 4-1 group I 0 -. 30 (100%) ' l lalobactedupi 0 26 (100%) • C■1 group II . • O 0 n •ri ird.,bacie,i o 8 000%) C.1 group II ° • + groupA cwe t obactcr IV 0 10 (100%) 0 0 miotococcux o ' 15(100%) 14 Z group V Arm,. Whitler 0 10(100%) group VI cs1 • Z group I o ' 33(100%) roup II 0 31 (100';,,) ; 0 Vibrio g roup III o 25(100S) i • 4.1 si':'. g roup IV 0 21(100%) o 4-1 roup V • mimp:J 1 group VI o 22(100'f,) cc) O )4 Total strains examined •CO 4-4e n. 610 It 18
a) Subgroup II: Germs forming colonies with irregular, slightly
convex edges, fimbriate, smooth, mobile with polar cilium, forming green-
brown pigments in King B medium with 7.5% of NaCl.
They oxidize the Hugh & Leifson medium and alkalize mediums with
other sugars. They use citrate, hydrolyze urea and.phenylalanine, they
do not hydrolyze tyrosine, they are lipolytic and gelatinolytic.
b) Subgroup III: Germs forming colonies with regular edges, smooth,
immobile, pigmented dark brown. The ST alkalize the Hugh & Leifson medium,
they use citrate, they hydrolyze phenylalanine, they do not hydrolyze
tyrosine, they are not lipolytic and they do not attack gelatine.
As regards the microbiological aspect of the hams, in table no. 3
we have reported the average values of the microbial contents after 10-15
days' immersion in brine.
The total microbial content showed average values of 600,000 and
consists almost exclusively by halotolerant germs.
In table no. 6, we have shown the classification of the bacterial
strains isolated from the muscular tissue of hams in the culture mediums
described. Furthermore, we evaluated (see the said table) their capacity
for reducing nitrates.
From an examination of table no. 6, we see that the germo which are
most represented are, in order of numerical importance, micrococci, sarcinae
and lactobacilli. Other groups of microorganisms are present in negligible
number.
Micrococci and sarcinae were found to be capable of reducing nitrates
to nitrites at 5 ° -8°C in the presence of 1-5% of NaC1, conditions which '
normally occur in salting. 19
Given the presence of a certain number of denitrifying halophiles in the brines, we wished to evaluate their significance and the role they play in successful curing.
To this effect, we experimented with two strains of denitrifying germs belonging to subgroups I (qicrococcus) and II (Halobacterium) which were the most numerous.
The cultures were started in three different mediums, at atmospheric pressure and under vacuum, at a temperature of 30°C and 5°C.
Working in on the basis of the data reported in tables 7, 8, 9, we also wished to determine if the capacity to reduce nitrates to nitrites was attributable to a respiratory process or to an assimilative process.
We can conclude that, for the strains examined, the process seems to be mainly of a respiratory type and can occur in aerobiosis and at a low oxygen concentration, at 30°C.
By lowering the temperature to 5°C and increasing the saline concentration, the microbial growth slows down and consequently the reduction of the nitrate and the nitrite diminishes considerably. Table 5
Differential characteristics of the sub-groups 1-4-5-6 which reduces nitrate to nitrite up to N2 + NO + NO2
Gram Appearance of the Respiratory Hydrolysis Attack on Production Attack Hydro Produc- staining colonies type (Hugh of the citrate of APP from on fats lysis tion of (*) & Leif son glycides phenyalanine of pigmen gelatine •
irregular edges oxide aerobes Micrococcus (fimbriate) subgroup I convex, . rough, brick red color
with irregular edges (fimbria- aerobes - A A Acinetobacter te) slightly subgroup IV convex, rough, red in color
with regular aerobes - A A edges, rounded, Micrococcus smooth, slightly subgroup V convex, white pigmented
with irregular aerobes - A A edges, rough, Acinetobacter variegated, quite subgroup VI convex, brick red _in color (*) For these descriptions the criteria of Cappellato and Narpozzi (1967) were followed. Legend: A = alkalinizers; APP = phenyl pyruvic acid. 21
Table 6
Classification of the bacterial strains isolated from the muscular tissue of raw cooking hams and their capacity ta'reduce nitrates to to N + NO + NO nitrites and 2 2 -
Number and percentage of strains Type of microorganisms which reduce nitrate to: -I- NO + NO NITRITE and N2 2
Halotolerant Sarcina spp. 23 (100%) 0 Micrococcus spp. 72 (100%) 0 Streptococcus D spp. 7 (100%) 0 + gram Microbacterium spp. ' 9 ( 0%) 0 Lactobacillus spp. 34 ( 0%) 0 Bacillus spp. 4 (100%) 0
gram - -Achromobacter-Alcaligenes 6 ( 60%) 0 phile Halo gram - ÇVibrio spp. 5 (100%) 0 Halobacterium spp. 8 (100%) 0
Total strains examined 178
Considerations and Conclusions
.The results rdported offer a very significant picture of the chemical
Structure and bacteriological aspect of the brines of cooking hams.
From an examination of the data on the sodium chloride. concentration
of the brines, it is noted that the values vary within very broad limits;
from 7.4 to 28.1%, while the average values are between 14 and 21%. This
wide variability in the data must be attributed'to the different methods of
preparation of the brines, some preferring the use of the so-called extremely
light'brines (8.5% NaC1), and others the use of light (12% NaC1), medium
(117 NaC1), strong (18% NaC1) and extremely strong (28% NaCl), brines. 22
As regards the pH, this also undergoes wide variations, ranging
from 6.2 to 8.5, with average values of 7-7.4. It is to be assumed that
the variations in the pH are due to the differing composition of the brines,
that is to say, dependent on the presence or absence of sugars, spices and,
probably, on the number of germs acting on the saccharose.
The total nitrogen values also show very wide variations, from
11 mg to 470 mg/100 ml, due, in our opinion, to the use of hams with
different aging times, having a different origin and subjected to a different method of storage.
The data relating to the amino nitrogen and to the free and fixed
ammonia are significant. The values of the amino nitrogen, although showing
broad variability, follow a particular trend in relation to the age of the
brine, in fact, the highest average values were found in 15-day brines; in
the 30-day brines, to the contrary, the mean values tend to diminish,
concomitant with an increase in free and fixed ammonia. This fact must be
attributed to the lysis of the soluble proteins by the active multiplying
microflora (see table no. 2) with the consequent increase of free amino
acids and, therefore of the amino nitrogen values. Table 7 Action on potassium nitrate of denitrifying halophile microorganisms .
Halobacterium strain in synthetic medium with 5% NaC1 at 30 °C
Atmospheric pressure Under vacuum
Time Nitrites NH PH 3 Growth pH Nitrites NH Growth h mg/100 ml N % CO % 3 N % CO % mg/100 in mg 2 2 in mg 2 2 mg/100 mg/I00 . of N of N2 ml of N 2 mi 2
_ — — — _ — _ — — _ _ — -- — — . , •,') . k 54 L25 40.1 LM M 6A 1.1 54 MA 46 . 702 0.i9 . '37 43 'k'.4 0.9 M8 . L69 H r 6A 13 P.(2J 6,4 0 297 2,13 68 ' 3 1 6,8 0.28 270 IA 1 65 35
Halobacterium strain 56 in medium with 2%0 extract of yeast with 5% NaC1 at 30° C
Atmospheric pressure Under vacuum
Microbial Time Nitrites NH CO pH Nitrites NH pH 3 wt N % % 3 Microbial h . mg/100 m1 gro h in 2 2 mg/100 — N % CO % tg/100 , of N mg/100 growth 2 2 mc of N ' 2 ml . 2 m1 mg
24 6.5 3.3 ' 215 -'.45 60 40 6A 0 • M.. 5.18 74 ' • 26 6A 71 3.96- M 6,6 ' 8.1 63 81 48 0 -)-7 69 0 —"6A 0 -, 3.65 75 M 65 0 • 8,1' 1134 80 . 20 1.2q 65 0 r 6:52 78 r 6,7 0 : 27 12,2 SO 20 ' • ' Ha1obacterium strains in medium with 2%0 extract of yeast, 4% NaCl at 30°C Atmospheric pressure Under vacuum
CO % Time pH Nitrites NH Microbial N % CO % pH Nitrites NH3 Microbial N2% 2 3 2 2 growth mg h mg/100 ml growth m g mg/100 . mg! 100 mg/100 ml ml ml _ .. -) •• 03 55 6.5 0.36 ' , 64.8 5.89 4S 6.6 13.5 2.-15 45 _ _ ls 6.6 0 151 6.58 '7l ' 29 6,6 ' 0 ' 162 ' ". _ 7' G ;,70 75 • 23 6,7 0 162 ..-. 199 .-,- ---, -.:, (•.6 0 6-23 ,:, ''') 65 0 170 8.%6 ., -,•. . M2 . . . _
Table 8 Action on potassium nitrate of denitrifying halophile microorganisms
, Strain 23 Micrococcus spp. in synthetic medium with 5% NaC1 at 30 ° C
Atmospheric pressure Under vacuum
al« Time Nitrites NT-T3 Microbial h pH N2% CO 2% pH Nitrites NHmg/100 Microbial N % CO % ml meloo growth mg 2 . 2 . 3 .2 2 mg/100 • mg/100 growth mi . of N ml mg 2 ml
. - 6A 0.62 - ,.= 0,23 21.6 1,1 75 25 0 162 -12 58 6. 1 0 2.3 ' 76 21 6.5 0 132 1.93 28 7'2 18,9 -,-- -,- 6,6 32.4 2,64 -, 0 -, 2.8 77 23 0 73
Strain 23 Micrococcus sp. in 2% yeast extract medium with 5% NaC1 at 30°C
Atmospheric pressure Under vacuum
Time pH Nitrites NH 3 Microbial Nitrites NH h mg/100 ml N % CO % pH Microbial N % CO % mg/100 growth 2 2 mg/100 3 2 2 ml mg mg/100 growth mg ml ml
0,36 54 5,98 6.5 0.59 54 55 ; 1 6.5 0 116 9,5 76 -, 6.5 0 71 8,8 75 25 O 73 6,6 75 2 , 6.6 0 92 8,5 60 40
Strain 23 Micrococcus sp. in 2% yeast extract medium 14% NaC1 at 30° C
Atmospheric pressure Under vacuum Nitrites NH Time pH 3 Microbial h mg/100 ml N % CO % • pH Nitrites NH3 Microbial mg/100 growth 2 2 • ml mg men° mg/100 growth N2% CO2 % ml ml mg 24 43 - - - 6,5 0,32 81 3,1' 64 36 6.4 - 72 6,5 o 156 0,7 43,2 1,9 70 30 62 78 22 6.6" O 96 6.6 0 135 7.6 78 22 135 7.4 66 34 N 120 6,6 o HO 72 e- 6.6 0 140 8,6 78 22 72 28 . 6,7 0 1.10 8 - - Table 9
Action on potassium nitrate of denitrifying halophile microorganisms
Strain 56 Halobacterium in medium with 2%dextract of yeast with 14% NaC1 at 5 ° C
Atmospheric pressure Under vacuum
Time Nitrates NH3 Microbial N % CO % pH Nitrites Microbial h PH mg/100 ml --3 2 2 NH N % CO % growth mg mg/100 mg/100 3 growth mg 2 2 ml ml mg/100 ml .
6,4 0 0 qnc qnc qnc 6,1 , 0,95 CUIC qnc qnc 2.4 7&,2 L5 97 3 6,7 3,4 70,2 qnc qnc
Strain 23 Micrococcus sp. in medium with 2% 0 extract of yeast with 14% NaC1 at 5 ° C
Atmospheric pressure - Under vacuum
Time pH Nitrites NH h 3 Microbial N% CO % pH Nitrites NH mg/100 ml 2 2 mg/I00 growth mg mg/100 3 Microbial N % CO % 2 2 ml ml mg/100 growth mg ml
2 r■ .-; 6,7 0,1 qnc qnc qnc 6,5 0,12 qnc qnc
4 56 6,7 qnc qnc Cirle 6,7 ; 0 qnc qr1C 26
In fact, all the germs isolated, produce ammonia from peptone.
The values of the nitrite concentrations are fairly constant in
the brines 5, 10, and 15 days old and vary between 12.4 and 14.6 mg/1; in
older brines, they diminish to the point of reaching average values of
mg 4.1/1 with variability limits between 4 and 10.5 mg/l.
The quantity of nitrites depends on various conditions: the dose
added by the preparer which does not always comply with the limits laid
down by law, the reducing capacity of the cellular and bacterial respiratory
enzymes, the extent of the microbial attack on the nitrates and nitrites,
the temperature which conditions the respiratory and multiplicative activity
of the germs, the pH.
Finally, we must mention the presence in the brines of lactic acid
in considerable quantity: in brines 30 days old, the average values found
are 321 mg/100 ml; with variability limits between 88 and 620 mg/l. The
presence of lactic acid is due to the enzymatic splitting of muscular
glycogen, with the consequent passage of the acid into the brine. The
longer the meat stays in the brine, the higher are the concentrations of
lactic acid: there are broad variability limits for this also: the
• concentration is determined by the factors already mentioned in connection
with the variations noted for the total nitrogen.
Thus summarizing, on the basis of the data obtained, it is possible
to have a general picture of the biochemical and microbiological processes
which occur in the brines, in spite of the marked variations found, which
are dependent on the various sampling conditions we were obliged to adopt. I , 27
The bacteriological examinations reported in tables 2, 3, 4, 5, 6
show the presence of a certain quantity of microorganisms.
The highest microbial concentrations are observed in the 15 and 30
day brines in which from 3 to 6 million germs per gram are counted. This
datum must be interpreted bearing in mind the ambient temperature (5-80C)
and the saline concentration. In this case also the variability limits are
very wide because the factors cited previously and others like the level
of contamination of the salt used, the initial microbial content of the
meat, spices, utensils, vats and of that brought about by handling during
curing have considerable effect on microbial multiplication.
The bacteriological analyses have revealed the presence of two
groups of microorganisms: the halotolerant and the halophiles.
The halotolerant group is represented by various microbial genera:
Sarcina, Micrococcus, Corynebacterium, Bacillus, Streptococcus, Lactobacillus,
(see table no. 4) Pseudomonas, Achromobacter, Alcaligenes.
The most numerically important microorganisms are those of the
Achromobacter-Alcaligenes group, the remainder are present in lesser quantity.
The group of halophile germs also consists of numerous microbial
genera.
We,must state in advance that a rigorous subdivision of these
microorganisms into genera is, at the present moment, premature because
their classification is still being worked out.
Many new species are described and many taxonomy problems must be
resolved before it is possible to arrive at a definitive schema of classifi-
cation. 28
For those who wish to study the problem more thoroughly, we would refer you to the works of Brisou (1955), Kriss (1961) ., of Kriss et al.
(1967), Oppenheiner (1966), Ferguson-Wood (1967), Drop & Ferguson-Wood
(1968) which deal extensively with the subject, to thé recent monograph by
Giannelli (1967), which has a copious bibliography and finally the most recent works of Psister et al. (1965), Monti et al. (1967), Simido & Hasuo
(1968 a, b), Johnson et al. (1968) Bohacek et al. (1968).
By following the criteria of the various authors and by bearing in mind the method of identification of the negative grams proposed by Shewan
(1966), we were able to identify microorganisms belonging to the genera
Sarcina, Micrococcus, Vibrio, Acinetobacter; numerous strains were temperarily grouped under the heading of unidentified Halobacterium.
Included under this term were coccoid-shaped and rod-shaped, gram negative and gram variable strains.
Over and above this traditional classification, for the purposes of our work, the halotolerant and halophile germs described may be subdivided into three groups according to their capacity for reducing nitrates to nitrites and to N2 + NO + NO2.
According to this criterion, the isolated halotolerant germs can be classified as: reducers of nitrates to nitrites and inert.
Belonging to the first group are all the strains of Sarcina,
Micrococcus Streptococcus, Pseudomonas and a large percentage (65%) of the
Achromobacter Alcaligenes. The Corynebacterium and the Lactobacillus were
shown to be inert. 29
As has already been stated, quantitatively, the most numerous germs are those belonging to the genus Achromobacter-Alcaligenes.
The halophile germs may be divided into three groups: inert, reducers of nitrates to nitrites, reducers of nitrites to N2 , NO, NO2 .
Among the reducers of nitrates to nitrites are the strains belonging to the genus Sarcina and Vibrio and 70% of those classified as unidentified halophile bacteria, 30% of which are inert.
Belonging to the group of bacteria reducing nitrate to N2 + NO + NO2 are strains of the genus Vibrio and Acinetobacter and others (table no. 4) named, on the basis of morphology, Halobacterium and Micrococcus.
Quantitatively, among the germs reducing nitrates to nitrites, the most numerous are those considered to be Halobacterium; less numerous are the
Vibrio and the Sarcinaè. The halophiles able to reduce nitrates to the end compounds N2 , NO, NO2 , never reach very high values, except for rare cases; when they are present, they constitute approximately 1-5% of the total flora.
The experiments, conducted to pinpoint the nitrate reduction process, essential to the formation of nitrosopigments which determine the red coloring of the meats, allowed us to establish that for this type of microorganism (at least within the limits of the strains examined) ; this is effected through a respiratory process and that this activity is strongly influenced by the concentration of sodium chloride and by the temperature.
On the basis of our tests, we consider that the use of saline concentrations lower than 8% NaC1 and temperatures higher than 8°C permits these germs to become active, with the consequent lowering of the nitrite concentration and therefore the possibility of obtaining Products of inferior quality. 30
In this connection, it is timely to call attention to the origin of halophile germs in brines: they most certainly come from the marine salt used, the degree of contamination of which has a determant effect on the composition of the microflora of the brines.
Before they are cooked, the microbial flora or the meat of hams
is different, since the most numerous germs are respectively Micrococci,
Lactobacilli, Sarcinae. Micrococci and Sarcinae are particularly active in reducing nitrates to nitrites which can also occur at 5°C in three days.
On the basis of the results obtained, it would appear to us that we can supply an overall picture of bacteriological conditions and the role played by the microorganisms during the curing (salting) period.
In the brines of cooking hams, the microbial flora can vary within certain qualitative and quantitative limits depending on the level and type of microflora present in the substances used.
The role played by the germs seems to consist mainly in the reduction of nitrate to nitrite while the formation of nitrosopigment appears mainly due to the activity of mitochondrial respiratory enzymes (Rose & Peterson,
1953), (Taylor & Walters, 1967).
The microbial genera responsible for the reduction of the nitrate are several: in our brines the halotolerant germs of the genus Achromobacter-
Alcaligenes and the halophiles of the genera Halobacterium and Vibrio play predominant role.
In meats, the reducing process is carried out principally by germs of the genus Micrococcus.
The presence of germs which accomplish complete reduction of nitrates and nitrites must be considered dependent on the quality of the mariue sait
and it has, in our opinion, an unfavourable effect on curing (salting). used #
The general process for curing (salting) meats through the use of
wet brines, outlined by Taylor & Walters, based on our experiments, can be
described schematically as follows:
NITRATE favourable . )Achromobacter microbial Alcoligenes › Halobacteriurn intervention ' Vibrio unfavourable 11ciobacterit, »1 _microbial I Acineobacter Micrococcus 'intervention - N + NO +.NO Ifibrio • 2 2 NITRITE (denitrifying germs) anaerobic activity of the cytochrome oxidase
C nitrosyl C ferrocytochrome ferrocytochrome
mitochondria 4- NADI-12 .... myoglobin .--. : . , muscular nitrite". metàmyo— metamyo;- mitochondria nitrosyl m9dglobin globin globin + ) NOMn Mb-oMb0 metMb nitrosyl 2 NO Met Mb 32
RÉSUMÉ
On reporte ici les renseignements analytiques sur la composition chimique et microbienne des saumures des jambons à ciire et la microbiologie des jambons à cûre. On décrit les germes alotolérants et alophils isolés. Leur rôle pour la salaison des jambons est ici d . :1 ir et examim:.
• SUMMARY • • Analytical data regarding chemical and bacteriological composition of cooked hams. Halotolerant and halophilic bacteria are described. Their role on curing is discussed and described. .• . • •
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BIBLIOGRAPHY
Italian bibliographic items
3. On the metering of nitrates and nitrites in stored meats. 11. Research on the microbial flora of dried salt beef brines. 12. Research on the flora reducing nitrates and nitrites in dried salt beef brines. 13. Studies on the aging of pressed neck of pork. 14. Description of heterofermenting halophile vibrions. 15. Elements of medical microbiology. 18. Research on the relationships between ammonia production and permeability of films in plastic material in Proteus Vulgaris. 19. The halophile vibrions of the brines for cooking hams. 27. The preservation of meats. 28. The products of deep frozen fish. 30. Notes on microbiological technique. 42. Research on the chemical composition of dried salt beef brines. 44. Influence of the temperature on the chemical and organoleptic properties of hams during the curing stage. 45. Observations on halophile microorganisms. 54. Notes on microbiological techniques.
German bibliographic items
1. The ammonia content of egg-white and egg-yolk. 18. Species and numbers of microorganisms in pickling brine. 38. Elementary pickling experiments. 39. Elementary pickling experiments, V. 40. Elementary pickling experiments, VI. 41. Elementary pickling experiments, VII. 42. Elementary pickling experiments, VIII, and conclusion.