This document discusses several non-lactic acid bacteria used as starter cultures in food fermentation and their properties, including Bifidobacterium, Propionibacterium, Brevibacterium, and Acetobacter. Bifidobacterium produces acetic and lactic acid and can grow from 25-45°C, with an optimal pH of 6.5-7.0. Propionibacterium is essential for flavor development in Swiss cheeses and grows internally from 25-32°C with an optimal pH of 6.5-7.0. Brevibacterium linens produces enzymes and compounds responsible for flavor and color in surface-ripened cheeses, growing aerobically from 20-

1. Other bacteria than
lactic acid bacteria
used as starter
cultures in food and
their properties
M. ANZA Adamou

2. introduction
– The fermentation is a bacterial process that takes place during the production
of numerous food products. It provides the final products with characteristic
aromas and textures and plays a crucial role in food safety and hygiene. Among
the bacteria responsible we have the lactic acid bacteria, which display high
morphological and physiological diversity and other non lactic acid bacteria
involved in the processes. We will present them as well as their caracteristics.

3. Bifidobacterium
– Bifidobacteria are indigenous inhabitants of the human intestinal tract, which is
one of the most complex microbial ecosystems (microbiota) on the planet,
comprising over 100 trillion bacteria in the large intestine (CABALLERO,
FINGLAS, & TOLDRA, 2016).
– Bifidobacterium is a Gram-positive, nonmotile, nonspore-forming, and
anaerobic organism. Bifidobacterial cells often stain irregularly with methylene
blue. Some species can tolerate oxygen, some are obligate anaerobes, and
some species can tolerate oxygen in the presence of carbon dioxide. These
organisms are catalase-negative; however, some species, such as
Bifidobacterium indicum and Bif. asteroides, possess weak catalase activity
when grown in the presence of air (Shah, 2011).

4. Bifidobacterium
– Bifidobacterium produces higher levels of acetic acid than lactic acid, usually in
the ratio of 3:2. These bacteria produce formic acid, ethanol, and succinic acid.
Some reports suggest that butyric acid and propionic acid are not produced.
However, studies in the author’s laboratory have shown the production of
butyric acid and hippuric acid by these microorganisms. Bifidobacterium can
grow in the temperature range of 25–45 °C, with a maximum growth
temperature of 43–45 °C and a minimum growth temperature of 25–28 °C. The
optimum temperature for growth of Bifidobacterium of human origin is
between 36 and 38 °C, whereas for those of animal origin this is between 41
and 43 °C. Growth of Bifidobacterium does not occur below 20 °C, and these
organ isms do not have thermoresistance above 46 °C (Shah, 2011).

5. Bifidobacterium
– The optimum pH for growth of Bifidobacterium is 6.5–7.0. No growth occurs
below the pH of 4.5–5.0 or above 8.0–8.5. Below pH 4.1, most species die in
less than a week even at 4 C, and below pH 2.5 most species die in less than 3 h.
Carbonate or bicarbonate can be readily used by Bifidobacterium as carbon
sources. However, Bifidobacterium cannot utilize fatty acids or organic acids as
carbon sources (Shah, 2011).
– The mean mol. % G + C of DNA for Bifidobacterium is about 58% (Batt &
Tortorello, 2014)

6. Bifidobacterium
– In the United States before the 1980s, the use of bifidobacteria in foods was limited to
a few products intended for therapeutic treatment. Among the earliest products was a
bifidus milk developed by Mayer in the 1940s for use in treatment of infants afflicted
with nutritional deficiencies. By the 1960s, enough evidence had been accumulated to
show it was possible to modify intestinal biota with B. bifidum. In the 1970s, Japan
produced its first bifidus product, a fermented milk containing B. longum and
Streptococcus thermophilus (in 1971). Bifidus yogurt followed in 1979. Growth of
bifidus foods and bifidus growth factor supplements continues to this day in Japan
with other countries of the world following suit. Products that have been formulated
with viable bifidobacteria and/or bifidus growth supplements include fermented and
nonfermented milks, buttermilk, yogurt, cheese, sour cream, dips and spreads, ice
cream, powdered milk, infant formula, cookies, candies, fruit juices, and frozen
desserts (Batt & Tortorello, 2014).

7. Propionibacterium
– Propionibacteria are pleomorphic rods, often diphtheroid or club shaped, but
may also exist as single cells, as pairs, or as branched cell aggregates; they are
anaerobic to aerotolerant and generally catalase-positive. The principal
propionibacteria associated with cheese are Propionibacterium freudenreichii, P.
thoenii, P. jensenii, and P. acidipropionici, often referred to as the dairy
propionic acid bacteria (PAB). Propionibacterium freudenreichii consists of two
subspecies, P. freudenreichii subsp. freudenreichii and P. freudenreichii subsp.
shermanii. The propionibacteria have temperature and pH growth optima at
25–32 C and 6.5–7.0, respectively. They are generally more sensitive to
conditions of high acidity than the lactic acid bacteria. Propionibacteria can
grow in the presence of 6–7% NaCl under optimum conditions, but at the low
pH found in cheese (pH 5.2–5.4) their growth rate in the presence of NaCl is
further reduced (Rattray & Eppert, 2011).

8. Propionibacterium
– Propionibacteria are essential for the development of the characteristic flavor
and eye formation in Swiss-type cheeses such as Emmental, Gruye `re, and
Appenzeller. Unlike P. camemberti, G. candidum, and B. linens, which grow on
the cheese surface, the propionibacteria grow internally in the cheese matrix.
The proteolytic activity of the dairy PAB is generally low, with a clear species
and strain variability. They grow poorly in milk, but addition of casein
hydrolysate to milk enables growth to significantly higher cell numbers (Rattray
& Eppert, 2011).

9. Brevibacterium
– Brevibacterium linens is a strictly aerobic microorganism with a rod–coccus
growth cycle, and has temperature and pH growth optima at 20–30 °C and 6.5–
8.5, respectively. Slow growth of this organism occurs in cheese-ripening
conditions, such as 12 °C and pH 5.5. It is a halotolerant microorganism, and can
grow in the presence of 15% NaCl. The growth of B. linens on the surface of
bacterial surface-ripened cheeses, such as Saint Paulin, Limburger,
and Münster, is preceded by the growth of yeasts and molds. The yeasts and
molds utilize the lactate present in the curd, and deacidification of the surface
occurs. This pH increase enables the growth of B. linens and other bacteria,
including B. casei, Arthrobacter spp., Corynebacterium spp., Micrococcus spp.,
and Staphylococcus spp (Rattray & Eppert, 2011).

10. Brevibacterium
– Brevibacterium linens produces extracellular aminopeptidases and proteinases,
the number and properties of which depend to a large extent on the strain. The
extracellular proteinases produced by B. linens are serine proteinases and are
highly active on αs1- and β-casein. In addition to these extracellular enzymes, the
presence of intracellular peptidases and proteinases has also been reported for
B. linens; however, these intracellular activities are low compared to the
extracellular activities. The production of extracellular lipolytic and esterolytic
activities by B. linens has not been determined unambiguously, with a number of
reports presenting conflicting data. However, intracellular esterases have been
detected and a number of them have been purified and characterized. One of
the most interesting and important properties from a cheese-ripening
perspective is the production of various volatile sulfur compounds, in particular
methanethiol, by B. linens (Rattray & Eppert, 2011).

11. Brevibacterium
– Brevibacterium linens is also characterized by its ability to produce various
bacteriocins and antimicrobial substances. The biochemical properties of the
bacteriocins produced by B. linens appear to be strain dependent, but at least
some of them have been shown to be inhibitory toward foodborne pathogens
such as Staphylococcus aureus and Listeria monocytogenes. Another important
property of B. linens is its unique yellow-orange aromatic carotenoid
pigmentation. The red-orange color of the surface of cheese varieties such as
Saint Paulin, Muünster, and Limburger is due primarily to the pigments
produced by Brevibacterium spp., Corynebacterium spp., Micrococcus spp., and
Arthrobacter spp (Rattray & Eppert, 2011).

12. Acetobacter
– Acetic acid bacteria (AAB) have been used for making vinegar at least since
Babylonian times. For most of this time, vinegar was obtained by fermentation
from natural alcoholic solutions (10–15% v/v ethanol) without an understanding
of the natural process. AAB belong to the family Acetobacter of the class
Acetobacteraceae. The family is classified into the former core genera,
Acetobacter and Gluconobacter, and eight genera. Species of Acetobacter (now
19 species) were partially newly classified, and a new genus was introduced,
Gluconacetobacter (16 species).
– Acetobacter are Gram-negative rods. Old cells may become Gram-variable. Cells
appear singly, in pairs, or in chains, and they are motile by peritrichous flagella
or nonmotile. There is no endospore formation (Hommel, 2014).

13. Acetobacter
– Acetobacter spp. are obligate aerobes except for Acetobacter diaztrophicus, for
example, which belongs to the diverse group of free-living aerobic or
microaerophilic diazotrophic AAB. Depending on growth substrates, some
strains may require p-aminobenzoic acid, niacin, thiamin, or pantothenic acid as
growth factors. The temperature range is 8–45 C with an optimum range
between 25 and 30 C. The optimal pH for growth is about pH 4–6.3. Acetophilic
strains have their optimum at pH 3.5, acetophobic ones at 6.5, and
acetotolerant strains can grow on both pH values. Strains used in making
vinegar are more resistant toward acidic pH values. Resistance is strain specific.
Isolates obtained from commercial submerged processes grow well at a pH of
2.0–2.3 (Hommel, 2014).

14. Different characteristics of the genera Acetobacter,
Gluconacetobacter, and Gluconobacter

15. Acetobacter
– Acetobacter spp. are used in different processes of making foods and food
additives. Vinegar is the most popular product of Acetobacter and
Gluconacetobacter made by incomplete oxidation. From the technical point of
view, one can differentiate between slow traditional and fast submerged
processes, respectively. In traditional vinegar making, AAB grow near/at the
surface where oxygen tension is high. Acetobacter spp. are involved in a
number of natural fermentations. A typical tropical beverage, palm wine, is
made from palm sap as a result of a mixed alcoholic, lactic acid, and acetic acid
fermentation by a complex microbial consortium. Acetobacter strains have also
been isolated from cocoa wine, made by fermentation of cocoa seeds (Hommel,
2014).

16. References
– Batt, C. A., & Tortorello, M. L. (2014). Encyclopedia of food microbiology Encyclopedia of food
microbiology.
– CABALLERO, B., FINGLAS, P. M., & TOLDRA, F. (2016). ENCYCLOPEDIA OF FOOD AND HEALTH. In B.
CABALLERO, P. M. FINGLAS, & F. TOLDRA (Eds.), ENCYCLOPEDIA OF FOOD AND HEALTH (pp. 4013).
– Hommel, R. K. (2014). Acetobacter A2 - Batt, Carl A. In M. L. Tortorello (Ed.), Encyclopedia of Food
Microbiology (Second Edition) (pp. 3-10). Oxford: Academic Press.
– Rattray, F. P., & Eppert, I. (2011). Cheese | Secondary Cultures A2 - Fuquay, John W Encyclopedia of
Dairy Sciences (Second Edition) (pp. 567-573). San Diego: Academic Press.
– Shah, N. P. (2011). BACTERIA, BENEFICIAL | Bifidobacterium spp.: Morphology and Physiology A2 -
Fuquay, John W Encyclopedia of Dairy Sciences (Second Edition) (pp. 381-387). San Diego: Academic
Press.