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Naturally fermented milk and its therapeutic

potential in the treatment of inflammatory


intestinal disorders
Cite as: AIP Conference Proceedings 2019, 060009 (2018); https://doi.org/10.1063/1.5061918
Published Online: 10 October 2018

Yoga Dwi Jatmiko, Gordon S. Howarth, and Mary D. Barton

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AIP Conference Proceedings 2019, 060009 (2018); https://doi.org/10.1063/1.5061918 2019, 060009

© 2018 Author(s).
Naturally Fermented Milk and its Therapeutic Potential in
the Treatment of Inflammatory Intestinal Disorders

Yoga Dwi Jatmiko1,a), Gordon S. Howarth2,3,b), Mary D. Barton4,c)

1
Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang, East Java,
Indonesia 65145.
2)School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy Campus, Roseworthy, South
Australia, Australia 5371
3)Centre for Paediatric and Adolescent Gastroenterology, Women’s and Children’s Hospital, Adelaide, South
Australia, Australia 5006
4)School of Pharmacy and Medical Sciences, The University of South Australia, City East Campus, Adelaide, South
Australia, Australia 5000.
a)
Corresponding author: jatmiko_yd@ub.ac.id
b)
gordon.howarth@adelaide.edu.au
c)
mary.barton@unisa.edu.au

Abstract. Naturally, fermented milk has become an essential commodity in rural areas in some developing countries,
especially in Africa and Asia. A variety of local products appear depending on local knowledge, including the type of
milk, containers, and techniques. Although the microbial composition of each product varies, lactic acid bacteria
commonly dominate the range of naturally fermented milk products. Research into functional benefits for human health
about the role of their microflora is rapidly expanding. The role of lactic acid bacteria as probiotics for either prevention
or treatment of human diseases has been a driving force in the exploration of new potential probiotics from naturally
fermented milk. Inflammatory intestinal diseases are one of the digestive problems caused by dysbiosis. Therefore, the
contribution of probiotics to improve the balance and diversity of intestinal microbiota has become a main concern. The
main focus of this review was to summarize a variety of naturally fermented milk products by highlighting the production
process and indigenous microflora involved. The determination of probiotics that are highly involved in the fermentation
of milk may have health benefits for treating inflammatory intestinal diseases, especially inflammatory bowel disease and
intestinal mucositis.

Keywords: fermented milk, microbial composition, microflora.

INTRODUCTION

Consumption of fermented milk products is prevalent in many countries due to their health benefits. Besides the
nutrition content of the milk itself, the health benefits are also determined by the activity of living microorganisms
(starter cultures) contained in the fermented milk.1 These microorganisms are responsible for improving the shelf-
life of the milk by increasing the acidity level and also the development of desirable organoleptic characteristics,
such as flavor and textures.2 Interestingly, some of the microorganisms, called probiotics, actively enhance human

The 9th International Conference on Global Resource Conservation (ICGRC) and AJI from Ritsumeikan University
AIP Conf. Proc. 2019, 060009-1–060009-16; https://doi.org/10.1063/1.5061918
Published by AIP Publishing. 978-0-7354-1737-3/$30.00

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health by improving the balance of intestinal microflora.3 Therefore, fermented milk products are considered to be
functional foods.4
Fermented milk products were initially produced by spontaneous fermentation using wild starter cultures, so-
called naturally fermented milk (NFM). Since the quality of final products is unpredictable, depending on the
dominant microorganisms initially involved, the production of fermented milk has been optimized utilizing
commercial starter cultures and controlled conditions.5 Yogurt is the most popular industrialized fermented milk
products. Through industrial production, the types of fermented milk products have become more diverse with
innovations such as the addition of probiotics, prebiotics, additive agents to enhance the organoleptic properties,
fresh fruits or even different milk types.5 Therefore, consumer preference has changed to these commercial products
rather than NFM products.6
In the era of industrialization of fermented milk, product development, as well as the issues of quality and safety,
have been thoroughly investigated. However, this approach could diminish the diversity of indigenous
microorganisms used and the organoleptic properties of NFMs.5-7 Some studies have shown that NFM products are
potential bioresources for the food technology industry, with starter cultures, probiotics, antimicrobial compound
and biocatalysts as examples of innovative end-use,6,8-13 Moreover, increased diversity of indigenous
microorganisms in the fermented milk can be expected to improve the balance and diversity of intestinal microflora.
In this current review, a variety of NFM products will be explored and their therapeutic potential, regarding
probiotics, in treating inflammatory intestinal diseases will be discussed.

Naturally Fermented Milk

Naturally fermented milk is a popular fermented product made by rural communities in some developing
countries. As part of the traditional heritage, a highly nutritious portion of milk is preserved through fermentation
that has significant therapeutic and social value (generating income).14 This product is prepared from fresh milk that
is fermented spontaneously without any inoculation of starter cultures.15 The art making of NFM products was
handed down from one generation to the next. NFM products are prepared by using unpasteurized or pasteurized
milk that is placed in a suitable container. After 2–3 days of incubation at an ambient temperature, the desirable
texture and flavor of the product will be achieved due to the role of indigenous microbiota.16, 17 NFM products are
regarded as small-scale products because they are manufactured at the household level in a range of communities in
some developing countries of Africa and Asia.15 As milk has a high nutritional value, natural fermentation is the best
way of enhancing the flavor and the nutritional benefits of milk as well as extending its shelf-life.17
A range of NFM products can be found around the world, each with a slightly different composition. Many
factors influence the unique characteristics of NFM products, such as the type of milk used, climate conditions
(temperature), pretreatment of the milk, fermentation conditions and the manufacturing process.18 As a result, the
local name of products is distinct by regions.17 However, in essence, NFM products have five principal similarities
among them.19 First, the indigenous microbiota initiating fermentation originates from the raw milk and the
environment, including the vessels and any equipment used. Consequently, a gradual selection of specific
indigenous microflora responsible for product stability and quality occurs, which is not easily imitated by modern
dairy starter cultures.16 Second, NFM products are manufactured by rural people with poor living conditions and
limited facilities. Third, a traditional and natural container is used, instead of plastic containers. Fourth, the
incubation condition is at ambient temperature for 2–3 days. Finally, the preparation of the products is under non-
aseptic conditions, without any knowledge about good manufacturing practices.
TABLE 1. Naturally-fermented milk products with identified microbiota and probiotics
Products Raw Country Microbiota Bacteriocin Probiotic Ref.
materials producers Strains
20, 21
Airag/ Mare’s Mongolia Lactococcus garvieae, Enterococcus -
Chigee milk Lc. lactis ssp. lactis, durans
Streptococcus
parauberis,
Enterococcus faecium,
Leuconostoc
mesenteroides and
Leuconostoc
pseudomesenteroides

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Products Raw Country Microbiota Bacteriocin Probiotic Ref.
materials producers Strains

14, 22
Amasi Cow’s South Lc. lactis subsp. lactis, - -
and mafi milk Africa, Leu. mesenteroides
Zimbabwe subsp. dextranicum,
Leu. citreum, Leu. lactis,
Lb. delbrueckii subsp.
lactis and Lb. plantarum
22-24
Amasi/ Cow’s Zimbabwe LAB (Lb. helveticus, Lb. Lb. plantarum -
hodzeko/ milk plantarum, Lb. AMA-K
mukaka delbrueckii subsp. lactis,
wakakora Lb.casei subsp. casei and
Lb. casei subsp.
pseudoplantarum)
Yeasts (Saccharomyces
cerevisiae, Candida
lusitaniae, C. colliculosa,
S. dairenensis, Dekera
bruxillensis, C.
lipolytica, C. tropicalis)
12, 13, 25-29
Dadih/ Buffalo West LAB (Streptococcus Lc.lactis Lb.
dadiah milk Sumatra, faecalis subsp. subsp. lactis plantarum
(yoghurt- Indonesia liquefaciens, S. cremoris, (IS-10285 & IS-10506,
like) S. lactis subsp. IS-16183), Lb. E.
diacetylactis, S. lactis, brevis IS- faeciumIS-
Leuconostoc 26958, Lb. 27526, Lb.
paramesenteroides, Lb. casei IS-7257 plantarum
casei subsp. casei and and Lb. S130
Lb. casei subsp. plantarum
rhamnosus, Lb. S130
paramesenteroides, Lb.
rhamnosus, Lb.
plantarum, Lb.
paracasei, Lb.
pseudomesenteroides,
Lc. lactis subsp. lactis);
yeasts (Endomyces lactis,
Pichia jadinii, Candida
stellimalicola), others
(Micrococcus varians,
Staphylococcus
saprophyticus, Bacillus
cereus var. mycoides and
Acetobacter cerevisiae)
11, 30-34
Dahi Cow’s India, Streptococcus bovis, Lb. Streptococcus Lb.
(yoghurt- and Pakistan fermentum, Lb. bovis J2 40-2, acidophilu
like) buffalo delbrueckii ssp. Lc. lactis CM1 sLA 02,
milk or bulgaricus, Lb. Lb.
mixture delbrueckii ssp. lactis, E. delbruecki
faecium, S. thermophilus, i subsp.
Leu. mesenteroides ssp. bulgaricus

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Products Raw Country Microbiota Bacteriocin Probiotic Ref.
materials producers Strains
mesenteroides, Leu. M3 40-3
mesenteroides ssp.
dextranicum, Lc. lactis
ssp. lactis, Lb.
raffinolactis and
Pediococcus.
Pentosaceus
35-38
Dangke Buffalo Makasar, LAB (Lactobacillus - -
(cheese- milk Indonesia spp.),
like)
9,39
Kule Cow’s Maasai, Lb. plantarum, Lb. - Lb.
naoto milk Kenya fermentum, Lb. paracasei acidophilu
and Lb. acidophilus s, Lb.
fermentum
40,41
Kurut Yak milk Qinghai, LAB (Lb. plantarum, Lb. - Lb.
China acidophilus, Lb. casei, acidophilu
Lb. fermentum, Lb. s E2, Lb.
brevis, Lb. minor, Lb. casei G12
curvatus), and yeasts
42
Raïb Cow’s Morocco Lc. lactis, Enterococcus Lactococcus -
milk faecium, E. faecalis lactis, E.
faecium, E.
faecalis
43,44
Rob Cow’s Sudan LAB (Lb. fermentum, Lb. - Lc. lactis,
milk acidophilus, Lc. lactis Lb.
and Streptococcus delbrecuki
salivarius); yeasts i, E.
(Saccharomyces faecium
cerevisiae and Candida
kefyr)
45
Sethemi Cow’s South LAB (lactobacilli, - -
milk Africa leuconostocs and
lactococci), yeasts
(Debaryomyces hansenii,
Saccharomyces
cerevisiae, Cryptococcus
curvatus, Cryptococcus
humicola and
Kluyveromyces
marxianus)
46
Shubat Camel China LAB (Lb. sakei, E. - -
milk faecium, Lb. helveticus,
Leu. lactis, E. faecalis,
Lb. brevis and Weissella
hellenica), yeasts
(Kluyveromyces

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Products Raw Country Microbiota Bacteriocin Probiotic Ref.
materials producers Strains
marxianus, Kazahtan
uiosporus and Candida
ethanolica).
47
Suusac Camel Kenya LAB (Lb. curvatus, Lb. - -
milk plantarum, Lb.
salivarius, Lc.
raffinolactis and Leu.
mesenteroides subsp.
mesenteroides); yeasts
(Candida krusei,
Geotrichum penicillatum
and Rhodotorula
mucilaginosa)
48,49
Tarag Cow’s Mongolia LAB (Lb. delbrueckii - Lb.
(yogurt- milk/cam subsp. bulgaricus, Lb. plantarum,
like) el milk/ helveticus, and S. Lb.
yak milk/ thermophilus), yeasts paracasei
goat milk (Kluyveromyces
marxianus,
Saccharomyces
cerevisiae, Issatchenkia
orientalis, and
Kazachstania unispora)
50
Gioddu Sheep or Sardinia Lb. paracasei, Lb. Lb. paracasei Lb.
goat milk plantarum and Lb. Olb46As2, reuteri,
reuteri Olb46As3, Lb.
and paracasei,
Olo45As2 Lb.
plantarum
51
Jben Cow’s or Morocco Lc. lactis Lc. lactis -
goat milk CCMM/IAV/
BK2
53
Sameel Sheep/ Saudi LAB (Lb. plantarum, Lb. - -
goat/ cow/ Arabia pentosus, Lc. lactis ssp
camel lactis, Lb. brevis, Lb.
milk salivarius, Lb. paracasei
ssp paracasei)
Yeast (C. lusitania,
Cryptococcus laurentii,
S. cerevisiae, C. kefy)
51-53
Koumiss Mare’s Central LAB (Lb. casei, Lb. Lb. plantarum Lb. casei
milk Asia fermentum, Lb. IMAU10116, Zhang, Lb.
(Mongolia plantarum, Lb. Lb. fermentum fermentum
, China) acidophilus, Lb. SM-7 SM-7
helventicus)
Yeast (C. pararugosa,
Dekkera anomala,

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Products Raw Country Microbiota Bacteriocin Probiotic Ref.
materials producers Strains
Geotricum sp.,
Issatchekia orientalis,
Kazachstania unispora,
Kluyveromyces
marxianus, Pichia
deserticola, P.
fermentans, P.
manshurica, P.
membranaefaciens, S.
cerevisiae, Torulaspora
delbruekii).
56
Nunu Cow’s LAB (Lb. fermentum, Lb.
milk plantarum, Leu.
mesenteroides, Lb.
helveticus, E. faecium, E.
italicus, Weisella
confusa, Lactococcus
spp.)
Yeast (C. parapsilosis,
C. rugosa, C. tropicalis,
Galactomyces
geotrichum, P.
kudriavzenii, S.
cerevisiae)
57
Chhu Caw’s India LAB (Lb. alimentarius, - -
milk, yak (Sikkim Lb. farciminis, Lb.
milk Himalaya) salivarius, Lb.
bifermentans, Lb. brevis,
Lc. lactis subsp.
cremoris)
Yeast (S. crataegensis,
C. castelli)

The NFM products listed in Table 1 can be divided into two groups based on the fermentation technique, namely
spontaneous fermentation and back-slopping. In spontaneous fermentation, the origin of indigenous microflora is
mainly from raw material (unpasteurized milk). The microbial succession is initially dominated by the growth of
LAB, followed by other microbes (yeast, mold and Gram-negative bacteria). In back-slopping, a small portion of a
previous fermentation product is then inoculated into the next batch of fermentation, which allows for faster
fermentation compared to spontaneous fermentation.58 In this review, NFM products will be categorized based on
their fermentation process.

Spontaneous Fermentation

Dadih

Dadih is a well-known naturally fermented milk product developed by local people in West Sumatra, Indonesia.
This product is manufactured using unpasteurized buffalo milk that is fermented spontaneously at an ambient
temperature of 13. This product is categorized as a yogurt-like product. The texture is smooth, soft and creamy.
However, sometimes the consistency is more robust and relatively dry, and this is determined by the content of
solids and lipids of the buffalo milk.59 Regarding raw materials and the preparation process, dadih is similar to dahi

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(NFM from Bangladesh and India), but the container used is different. Dahi is prepared in earthenware pots 30, while
dadih utilizes bamboo tubes.13 A study of the chemical composition on dadih showed that the pH is approximately
3.4, water content 84.35%, protein 5.93%, fat 5.42%, and carbohydrate 3.34%.60 The quality of buffalo milk
influences the chemical composition in each product.
The preparation process of dadih has three main steps. First, fresh buffalo milk is filtered and poured into a fresh
bamboo tube. Second, the bamboo tube containing buffalo milk is covered with banana leaves, previously wilted by
heating. Last, incubation is conducted at ambient temperature for 2–3 days.61 The shelf-life of dadih is three days at
ambient temperature; beyond this day, the dadih surface will turn yellow and undesirable microorganisms will grow,
such as fungi.62

Dangke

Another important NFM product in Indonesia is dangke, which originates from Enrekang Regency, Province of
South Sulawesi. This product is prepared from heat-treated buffalo milk, and then processed enzymatically utilizing
papain from papaya latex .36 Thus, Surono et al. 58 classified dangke as a cheese-like product. The manufacturing
process of dangke is not as simple as dadih. To begin, freshly drawn buffalo milk is heated until boiling. After
cooling to about 90oC, papaya (Carica papaya) latex from the sliced leaves and the unripe/young fruits is added as
required to the warm buffalo milk. Papaya latex contains a papain enzyme that plays an important role in
coagulating the milk proteins. The addition of the papaya latex should not be excessive because a strong bitter taste
results. When the milk containing the papaya latex is stirred gently, a separation between curd (solid) and whey
(liquid) is formed. The curd is filtered and poured into a coconut shell, prepared with a small hole at the bottom for
draining off the whey. Then, the curd is pressed to separate the excess whey. Finally, curd with an arch shape is
detached from the coconut shell, wrapped with banana leaves and kept at room temperature until ready for
consumption. The curd, usually, is immersed in a salt solution overnight before wrapping to extend the shelf-life.59
Dangke is the only cheese-like product using a coagulant derived from higher plants and also using heat-treated
milk. It has been assumed that heat treatment associated with the protein coagulation determines the product’s taste.
Surono et al.59 confirmed that the higher the temperature (~ 90oC), the greater activity of milk clotting, whilst the
proteolytic activity decreased. As a result, a strong bitter taste is not developed in dangke.

Tarag

As part of an ancient diet in the Mongolian community in China, tarag is a spontaneously fermented milk
product made from milk of domesticated livestock, such as cow, yak, goat or camel. 48 In Zang and Chaidamu
regions, tarag is commonly made from goat milk. The production process of tarag in these regions is based on
traditional a technique in which the whole raw goat milk was poured into a large leather bag that is then tied. The
milk is then fermented naturally at temperatures of 15–20oC for at least two days.63 The general characteristic of the
final product is recognized by alcoholic flavor and sour taste. Zhang et al. 63 also showed that tarag has a higher
nutritional content as well as a high number of indigenous microflora, especially LAB and yeast. The diversity of
LAB in tarag is very abundant as shown by Liu et al. 64, with the predominant species being Lactobacillus
helveticus, Lactococcus lactis subsp. lactis and Lactobacillus casei. Another study revealed that Lactobacillus
delbrueckii subsp. bulgaricus, L. helveticus and Streptococcus thermophilus are the predominant LAB species, while
Saccharomyces cerevisiae, Issatchenkia orientalis, and Kazachstania unispora are the predominant yeast species.48

Amasi

Amasi, also called hodzeko or mukaka wakakora, is traditional fermented cow’s milk produced in Zimbabwe,
South Africa, Lesotho and other countries in Southern Africa.65 This product is made by fermenting raw cow’s milk
spontaneously in an earthenware (clay) pot or gourd (calabash) for two or three days at ambient temperature. 66 After
the whey is drained, curd with a thicker texture than yogurt is the expected final product, characterized with a mildly
sour taste (pH between 3.6 and 4.2) and aromatic flavor.66,67 A microbiological study of this product detected the
presence of fermentative microbes (LAB, yeast) and spoilage microflora (coliform, mold).68 Three LAB genera
dominate this product: Leuconostoc (Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc citreum and

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Leuconostoc lactis), Lactococcus (Lactococcus lactis subsp. lactis) and Lactobacillus (Lactobacillus delbrueckii
subsp. lactis and Lactobacillus plantarum).14 A bacteriocin was also found, called AMA-K and produced by
Lactobacillus plantarum.66 The yeast population was dominated by Saccharomyces cerevisiae, Candida lusitaniae,
Candida colliculosa and Saccharomyces dairenensis.67

Kule naoto

Kule naoto is a spontaneously fermented cow’s milk product produced in the Maasai community, Kenya. 39
Unpasteurized cow’s milk is fermented in a particular container (wooden gourd) made from the dried fruit of
Lagenaria siceraria at ambient temperature for five days or even longer until the desired texture, flavor and aroma is
achieved. A special pretreatment of the container differentiates this product from with other products regarding its
production process. The pretreatment applied is an addition of fresh blood from a cow, and the inside of the gourd is
rubbed with charcoal of a stick from the tree of Olea africana.9 The consistency of the final product is similar to
yogurt or fresh cheese; however, the taste and aroma of kule naoto seem more natural when the acidity level is not
more than 4.5. 39 The fermentative LAB isolated were Lactobacillus plantarum, Lactobacillus fermentum,
Lactobacillus paracasei and Lactobacillus acidophilus, with L. plantarum in the highest numbers.39

Kurut

Kurut, a popular product in the Qinghai-Tibetan highlands, China, is prepared from raw yak milk fermented
naturally in a big container (tung-made jar) at ambient temperatures of around 10–15oC for 7–8 days.40 In addition to
direct consumption, kurut is commonly also further processed to derivative products, such as butter (mar) and
cheese (cula).69 The organoleptic characteristic of this product is similar to kefir or koumiss with acidic taste and
alcoholic flavor.40 The presence of lactic acid is a result of fermentative LAB activity with Lactobacillus fermentum
and Lactobacillus casei as predominant isolates.70 Another study has shown that Lactobacillus delbrueckii subsp.
bulgaricus and Streptococcus thermophilus were the most frequently isolated.71 The occurrence of yeast has been
studied thoroughly, which determined Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia fermentans
as the predominant species.72

Raib and jben

Raib and jben. Raib and jben are traditional Moroccan fermented milk products, a type of yogurt and cheese
product, respectively.73 Although cow’s milk is a common raw material, other milk from sheep, goat and camel also
can be used. These products are made by fermenting raw milk spontaneously at ambient temperatures of around 15–
25oC for 1–3 days, with the fermentation period being affected by season.74 The curd or coagulated milk is called
raib. The coagulated milk is then placed into a cloth for draining its whey. Then, salt is added to this dehydrated
product to make jben.73,74 The acidity level of raib and jben, respectively, is 4.2 and 4.1, while lactic acid content is
0.67 and 1.04%, respectively.73 Three LAB groups were observed to be predominant in both raib and jben, namely
streptococci, lactobacilli and leuconostoc. In raib, Streptococcus was the dominant genus, while in jben the number
of these three genera were found equally.73 Like other NFM products, yeast was also detected in both raib and jben.
However, coliform was also found, reflecting poor sanitation during preparation of the products.73 The existence of
antimicrobial-producing LAB isolates from raib has been evaluated. Five presumptive bacteriocin producers having
high antimicrobial activity against Listeria monocytogenes and Staphylococcus aureus was found.75 In another study
of raib, anti-Listeria monocytogenes activity was also exhibited by Lactococcus lactis, Enterococcus faecium and
Enterococcus faecalis.76

Sethemi

Sethemi, South African naturally fermented milk, is prepared by fermenting unpasteurized milk spontaneously in
a gourd or clay container at ambient temperature with final acidity levels around 4.1–4.3.77 Even though information
for LAB identified from this product is not available, the interaction between LAB and yeast has been studied.
Kebede et al. 77 reported that LAB growth was enhanced in the presence of Saccharomyces cerevisiae at a
temperature of 25oC.

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Suusac

Besides kule naoto, Kenya is also known for fermented camel’s milk called suusac. Suusac is a NFM product
prepared from unheated camel milk using a spontaneous fermentation technique. The use of pretreatment (before
fermentation starts) the gourd is also a distinguishing technique. In suusac, the gourd is first treated with smoke.
Hence, after 1–2 days of fermentation at ambient temperature (26–29oC), the final product is characterized by white
color and less-creamy texture, with smoky flavor and a sharp acidic taste 46. The existence of LAB and yeast
interactions in developing this product was investigated by Lore, Mbugua and Wangoh.47 The most frequently
isolated LAB and yeast species were Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus plantarum
as well as Candida krusei, respectively.47

Nunu

Nunu. Nunu is prepared from unpasteurized cow’s milk produced by spontaneous fermentation. This product is
made by rural communities (Fulani migrants) in Ghana and some areas in West Africa. 78 The fermentation
technology applied in this product is quite simple. Cow’s milk is stored in calabashes, gourds, clay pots or plastic
containers, and then incubated at ambient temperature for 1–2 days.79 After churning the product, some whey and
butter/fat is separated to obtain the curd.78 In Nigeria, the whey and butter/fat is not removed, and it is called nono.80
A standard way to consume these fermented products is served alone or with fura (fermented pearl millet based
product).78,79,81 A recent study showed that Lactobacillus fermentum was the most frequently isolated LAB species
throughout the fermentation period. The other detected LAB species were Lactobacillus plantarum, Leuconostoc
mesenteroides, Lactobacillus helveticus, Enterococcus faecium, Enterococcus italicus, Weissella confusa and
Lactococcus spp..78 Yeast isolates identified in this study were Candida parapsilosis, Candida rugosa, Candida
tropicalis, Galactomyces geotrichum, Pichia kudriavzevii and Saccharomyces cerevisiae with P. kudriavzevii and S.
cerevisiae as the predominant species.78 Although some coliform bacteria were detected in nunu, at the end of
fermentation they were not detected anymore, reflecting the inhibitory effect of low pH.79

Back-Slopping Fermentation

Dahi

Dahi is a famous traditional fermented milk product from South Asian countries, especially India, Pakistan and
Bangladesh.11,82,83 The milk used in this product can be obtained either from a cow, buffalo, goat or a combination of
cow and buffalo milk 81. Dahi is considered a yogurt like-product. However, dahi and yogurt are different regarding
flavor. Dahi flavor is a result of diacetyl metabolite activity of mixed mesophilic lactococci strains. In contrast,
acetaldehyde flavor is the main characteristic of yogurt.84 The uniqueness of traditional fermented milk products is
mainly determined by the production process.
The back slopping technique is applied in the production of dahi. The process is started by heating milk until it
reaches the boiling point or the milk volume decreases to 15–20%. After the temperature is similar to body
temperature, 2–3% starter (previous Dahi product) is added and then poured into an earthenware vessel. A woolen
cloth or a straw or jute bag is used to wrap the vessel, and it is kept at ambient temperature overnight until the curd
is formed. The appearance of dahi can be distinguished by its solid texture, brown color and caramelized flavor. 83,85
Indigenous microflora isolated from dahi are dominated by lactic acid bacteria. Harun-ur-Rashid et al. 83 reported
that Streptococcus bovis was the predominant strain. In a review, Kumar et al.86 reported that some LAB members
found in dahi were Streptococcus, Lactobacillus, Pediococcus and Weissella. The presence of the genus
Lactococcus was also reported as the predominant strain.87 Standardization of the dahi preparation process was
initiated by the Bureau of Indian Standards (BIS). One of the standards implemented is the use of a starter
containing Lactococcus lactis subsp. Lactis and L. lactis subsp. diacetylactis or these cultures mixed with
Leuconostoc species.82
In some regions in the highlands of Himalaya, dahi is further processed to be a number of other fermented milk
products, such as gheu, mohi, soft chhurpi and chhu. The making process of these fermented derivative products is

060009-9
more complicated than dahi preparation. Take chhu as an example. Chhu is an indigenous fermented cheese-like
product, and its preparation process has been explained by Dewan and Tamang 57 and Tamang et al. 88. After dahi is
obtained, it is churned in bamboo or wooden vessel with addition warm or cold water to get butter-milk. Then, the
butter-milk is cooked until a soft and white mass results. After this, the remaining whey contained in the soft mass is
drained by placing the soft mass into a muslin cloth. This soft mass is fermented further in a closed container at
ambient temperature for 5–7 days to obtain fresh chhu. The identified LAB species from chhu were Lactobacillus
alimentarius, Lactobacillus farciminis, Lactobacillus salivarius, Lactobacillus bifermentans, Lactobacillus brevis
and Lactococcus lactis; yeast was identified as well (Saccharomycopsis catagenesis and Candida castelli).57

Airag

Airag is NFM product derived from unpasteurized mare or camel milk fermented using previous airag (back-
slopping). Airag, also called koumiss, is well-known fermented milk in Mongolia, Kazakhstan, Kyrgyzstan and
some Central Asian regions of Russia.48 The development of traditional fermented milk products from those
countries was influenced by Mongolian nomadic people, who prepared the fermented product using natural tools
and ingredients.49 The organoleptic characteristic of airag is a mild alcoholic flavor with acidic taste. 48 This
particular characteristic is a result of interactions between LAB and yeast. Sudun et al. 89 reported that yeast growth
and its ethanol production was enhanced when co-culture between yeast and LAB species isolated from airag was
conducted. Microbial composition of airag was dominated by LAB strains, namely Lactobacillus helveticus and
Lactobacillus kefiranofaciens, with Kluyveromyces marxianus as the predominant yeast.48 Watanabe et al. 90 also
successfully isolated a new Bifidobacteria species from airag and proposed with the name Bifidobacteria
mongoliense sp. nov.

Rob

Rob is another back-slopping fermented milk product produced in Sudan. This product is produced as a result of
milk surplus after a Sudanese family has consumed enough. After an overnight fermentation at ambient temperature,
the fermented milk is then churned in a tanned goatskin (Si’in) or a special-made container called Bukhsa from the
dried fruit Lagenaria peucantha. 43

Shubat

Shubat is a traditional fermented camel’s milk that is not only widely consumed in Kazakhstan, but also in
Mongolia, Uzbekistan, Turkmenistan and some areas in Russia. 91 Shubat is made using a back-slopping technique,
in which a small amount of shubat is left in the container, and fresh camel’s milk is poured on top to make another
fresh product.92 Unlike typical yogurt, shubat is in liquid form instead of a creamy form. Additionally, CO 2 gas is
produced, and the pH is around 3.8.92 In Kazakhstan, medicinal effects of camel milk are highly appreciated by
consumers, mainly to assist in the treatment of tuberculosis patients. As a result, large-scale production of shubat has
grown.92 Microbial flora was assessed using a polyphasic approach by Rahman et al 92 They identified some
important LAB species in shubat from the genera Lactobacillus, Enterococcus, Lactobacillus, Leuconstoc,
Enterococcus, and Weissella, with Lactobacillus sakei and Enterococcus faecium as the predominant species.
Kluyveromyces maxius was reported as the dominant yeast.92

Health Properties of Naturally Fermented Milk

Milk, which constitutes a highly nutritive component of the healthy human diet, is perishable and susceptible to
contamination. A detailed description of bioactive components in milk was reviewed by Ebringer et al. 94 It includes
certain vitamins, specific proteins, bioactive peptides, oligosaccharides and organic acids. Natural fermentation of
milk is principally aimed at extending the shelf-life of the product.3 Indigenous microbiota plays an essential role in
the fermentation process by producing metabolite products, such as organic acids, amino acids, bioactive peptides
and enzymes.95 Accordingly, the value added to milk is increased, and NFM products are, in turn, categorized as
functional foods.94 The predicate of functional food is also strengthened by the presence of probiotics as the main
contributor to the health properties of NFM products.8 For this reason, the research area of NFM products is

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significantly increased by exploring the role of their microbiota in traditional fermented milk products from many
regions.
The benefits of NFM products are mainly attributable to the microbial flora, which are not only responsible for
the organoleptic development but also play an important role in the therapeutic activity, such as improving digestive
properties as well as anti-diarrheal and antimicrobial properties. Therefore, the benefits of fermented products are
classified into two areas, namely nutritional and safety benefits.96 In nutritional benefits, the role of microbial
enzymes is predominant. The enzymes can transform substrates so that they become more digestible. For instance,
the lactase (galactosidase) enzyme in fermented milk products can improve lactose intolerance problems in
humans.96 Amino acids and vitamins are two essential health products generated by microbes, which provide
additional value to fermented products.97 Also, to improve the nutritional value of fermented milk products, genomic
and proteomic techniques can be applied to accurately define the function of enzymes for specific nutraceutical
applications.98 In fact, the NFM products are a potential functional food source because they contain useful
substances, which requires further exploration.99
The nutritional composition of NFM products has been widely investigated worldwide. Tarag, traditional
fermented goat milk from Mongolia, has higher nutritional value when compared to yogurt. The beneficial
constituents are casein, lactoferrin, serum albumin, β-lactoglobulin and α-lactalbumin.100 A similar composition was
also observed in kurut (naturally fermented yak milk) from Qinghai, China 39. Dahi (naturally fermented buffalo
milk) from India and Bangladesh contains protein (22.5% DM (dry matter)), fat (24.5% DM) and carbohydrate
(48.2% DM).101 Although the nutritional information available is not explicitly related to the functional properties,
dahi is considered to assist in amelioration of intestinal diseases, such as constipation, diarrhea and dysentery.30
Furthermore, it has been found that milk contains bioactive peptides.102 Angiotensin I-converting enzyme (ACE)
inhibitors have been intensely studied recently as bioactive peptides. The active peptide is known as an anti-
hypertensive peptide because its activity can reduce peripheral blood pressure by inhibiting ACE activity. 103
Koumiss, a traditional fermented mare’s milk, has been reported to be rich in ACE-inhibitory peptides.104 Besides
ACE-inhibitory activity, NFM from Tibet (yak and cattle milk) had a higher concentration of Y-aminobutyric acid
(GABA), which is also suitable for hypertension treatment.105
The second benefit of fermented products is food safety. Lactic acid bacteria are the microbial group commonly
dominating naturally fermented milk products.106, 107 This group of bacteria can produce some metabolite products,
such as organic acids, hydrogen peroxide, carbon dioxide, diacetyl, and bacteriocin, which have an antibiosis effect
against undesirable microorganisms 95. These compounds can inhibit the growth of some spoilage and pathogenic
microbes. These microbes might be found in NFM products because the preparation process is characterized by poor
hygienic conditions.36 Moreover, antimicrobial substances, especially bacteriocins, have been applied as food
preservatives as well as an alternative to antibiotics for treating infectious diseases. 108, 109

Naturally, Fermented Milk Products Improve the Diversity of Intestinal Microbiota

Human intestinal microflora plays an essential role in disease and health. The existence of intestinal microflora
contributes not only a negative impact (source of infection) but also a positive effect (protection against diseases and
maintenance of intestinal function).110 Increasing the number of beneficial microbiota in the intestines is the purpose
of probiotic supplementation. According to Tuohy et al.110, bifidobacteria and lactobacilli are the most essential
components of beneficial intestinal microbiota. As functional foods, NFM products may contain probiotics that
contribute to modulate intestinal microbial composition, especially increasing beneficial microflora.
Functional properties of NFM products are not only shown by the presence of essential compounds but also the
existence of viable microbiota, called probiotics. As mentioned earlier, LAB and yeast or combinations of these are
responsible for the natural fermentation of milk.107 There has been much discussion about LAB as probiotics. 111
However, probiotic microorganisms are not always LAB members, such as Lactobacillus, Leuconostoc, and
Streptococcus. Probiotic properties are also demonstrated by yeasts (e.g., Saccharomyces strains) from fermented
food products.28, 112 Therefore, there is a growing interest to investigate probiotics from fermented milk products,
even from non-dairy products.37,113
Many commercial probiotic strains (bifidobacteria and lactobacilli) are generally enteric flora. Interestingly,
recent studies showed that microorganisms isolated from NFM products also show probiotic characteristics (Table
1). Among the probiotics obtained from a wide range of NFM products, only Lactobacillus casei Zhang from
koumiss, China has been comprehensively tested and patented.55 Besides its probiotic properties, this bacteria
demonstrated other beneficial health effects, namely immunomodulation,35 antioxidative effects114 and

060009-11
antihypertensive activity.115 Also, another probiotic derived from koumiss was Lactobacillus fermentum SM-7,
which was able to reduce cholesterol levels both in vitro and in vivo (using mice).53 In clinical studies, a positive
effect of Enterococcus faecium IS-27526 (probiotic from dadih, naturally fermented buffalo milk of Indonesia)
against humoral immune responses and bodyweight increase on preschool children was observed using a
randomized double-blind, placebo-controlled study.12 The probiotics mentioned before are evidence that NFM
products could be a rich source of probiotics.
Dairy products are the most suitable matrices for a probiotic carrier as well as a natural habitat. The main reason
is milk, and its derivative products are an environment with the high nutritive value required for wild-type bacterial
growth, especially LAB.113 In NFM manufacturing, the indigenous LAB occupies a central role in spontaneous
acidification of the raw milk in the absence of added commercial starter cultures.18 Due to their nutritional contents,
NFM products are considered to be a complex ecosystem with a variety of metabolic activities produced by LAB
and non-starter LAB during fermentation. This condition leads to the indigenous LAB withstanding competition
from other microorganisms by producing antimicrobial substances and other supporting compounds.116 The presence
of bacteriocins (antimicrobial peptides) has been reported from some NFM products as can be seen in Table 1.
Furthermore, NFM products are also a rich source of either essential nutrients or beneficial microflora compared to
conventional yogurt, as shown in tarag. Zhang et al.63 highlighted that tarag had a higher content of casein,
lactoferrin, serum albumin, β-lactoglobulin, α-lactalbumin, vitamins and minerals, and had a more significant
number of LAB and yeast than yogurt. Hence, isolation of probiotic candidates from NFM products can be used to
increase the availability of probiotics with unique characteristics.

The rationale for the use of Probiotics for Treating Inflammatory Intestinal Disorders
Inflammatory bowel disease (IBD) and intestinal mucositis, two inflammatory intestinal disorders, share many
similarities concerning pathogenesis. A disturbance of homeostasis of the gut microbiota, epithelial cell disruption,
inflammation, microbial infection and immune response dysfunction are acknowledged as contributory factors to the
development of both IBD and intestinal mucositis.117 Effective therapeutic strategies should consider improvements
in the balance of gut microbiota, increase the ability of microbiota to suppress the inflammatory response, increase
epithelial cell integrity and increase immune functions. The use of probiotics and their products have shown
beneficial effects to improve intestinal health through these modes of action, which are principally applicable to both
diseases.118
Accumulating research suggests that probiotic therapy offers a prospective strategy to reduce the severity of IBD
and intestinal mucositis and restore the gut microbiota balance. Probiotic microorganisms for IBD therapy have been
used in clinical trials, such as Saccharomyces boulardii, Lactobacillus GG, E. coli Nisle 1917 and VSL#3.119 A
recent investigation showed that Lactobacillus reuteri BR11 could prevent the pathogenesis of IBD in experimental
colitis in rats, which was contributed to the cysteine-uptake system.120 Similarly, the use of Lactobacillus fermentum
BR11 was able to normalize colonic damage in the treatment of dextran sulfate sodium (DSS)-induced colitis.121
Investigation of probiotics as potential treatments of intestinal mucositis is limited compared to IBD.
Additionally, successful application of probiotics in treating chemotherapy-induced intestinal mucositis has not been
demonstrated yet. For example, potential beneficial effects of Lactobacillus fermentum BR11, Lactobacillus
rhamnosus GG, and Bifidobacterium lactis BB12 were not demonstrated to treat chemotherapy-damaged small
intestine.122 The investigators recommended further study using a higher dosage, timing variations and mixture of
probiotic cultures. However, administration of probiotic supernatants presents the possibility of adjunctive therapy
for intestinal mucositis. Supernatants of Lactobacillus fermentum BR11, E. coli Niisle 1917 and Faecalibacterium
prausnitzii showed partial beneficial effects to prevent intestinal damage due to 5-fluorouracil (5-FU) regimen.123,124
Therefore, further studies are still needed either to justify recommendations based on previous research or to explore
novel probiotic candidates from potential sources.
Because mechanisms of action of probiotics are strain specific,125 prospective probiotic candidates are urgently
required, especially those specific for inflammatory intestinal diseases. Although the most commonly used
probiotics include intestinal strains of Lactobacillus and Bifidobacterium species, other intestinal microbes or
microbes from other ecological niches may also have a beneficial role in human health.3 Therefore, NFM products
hold great potential as prospects for rich resources of probiotics with potential health-improving properties.

060009-12
SUMMARY

Naturally fermented milk products as a part of indigenous knowledge are not only meaningful for future
therapeutic development but also give insight into the importance of health benefits within the context of probiotics.
The capability of probiotics in treating inflammatory intestinal disorders has been elucidated in many studies;
however, exploration of probiotics from NFM products against this sort of disease considerably limited.
Furthermore, more multidisciplinary research should be conducted as more effort is required to educate rural
communities to produce NFM products with better safety and quality as part of preventive actions against
inflammatory intestinal disorders.

ACKNOWLEDGMENTS
We would like to express my deepest gratitude to the 9th ICGRC 2018 committees for accepting this manuscript
to be published in this proceeding.

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