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Pustaka I Jenis-jenis BAL yang terdapat pada kambing etawa

Artikel I

Fitri, Indah Nur dan Tri Ardyati, 2014. Skrining Bakteri Asam Laktat asal Susu Kambing
Peranakan Etawa sebagai Penghasil Bakteriosin. Jurnal Biotropika, Volume 2, Nomor 3,
Halaman 164-168.

Impact Factor: 1.944

Dari artikel ini didapatkan metode isolasi dan identifikasi bakteri asam laktat asal susu kambing
etawa serta hasil dari isolasi bakteri asam laktatnya.

A. Metode Isolasi dan Karakterisasi Bakteri Asam

Sebanyak 25 ml sampel susu kambing PE dimasukkan ke dalam 225 ml NaCl 0,85 % untuk
memperoleh seri pengenceran 10-1. Pengenceran bertingkat dilakukan hingga 10-6. Bakteri asam
laktat diisolasi menggunakan media de Man, Rogosa and Sharpe agar (MRS) yang mengandung
1% CaCO3 dengan metode pour plate. Isolat BAL yang tumbuh dicirikan oleh adanya zona
bening dan dimurnikan hingga diperoleh koloni tunggal dan ditumbuhkan pada media MRS agar
miring sebagai stok. Isolat BAL dikarakterisasi dengan pengamatan morfologi koloni, uji
katalase dan pewarnaan Gram.

B. Identifikasi Isolat Bakteri Asam Laktat dengan API 50 CHL Test Kit

Sebanyak dua ose isolat BAL yang telah berumur 84 jam diinokulasikan ke dalam 5 ml
aquades steril. Selanjutnya suspensi tersebut diinokulasikan sebanyak 2 ml ke dalam 5 ml
aquades steril hingga kekeruhan mencapai 2 McFarland. Sebanyak 4 ml larutan pada kekeruhan
2 McFarland diinokulasikan ke dalam ampul yang berisi medium API 50 CHL dan
dihomogenasi. Isolat BAL yang terdapat pada medium API 50 CHL diambil dengan mikropipet
steril dan diinokulasikan ke dalam strip yang telah berisi media karbohidrat kemudian ditutup
dengan mineral oil sebanyak dua tetes pada masing-masing strip. Selanjutnya strip media API 50
CHL tersebut diinkubasi pada 37C selama 48 jam. Pengamatan dilakukan dengan mencatat hasil
uji positif atau negatif pada setiap strip. Hasil positif ditandai dengan berubahnya warna reagen
menjadi berwarna kuning dan khusus untuk strip nomor 25 berwarna hitam. Hasil negatif
ditandai dengan tidak adanya perubahan warna pada strip (warna biru keunguan). Hasil
pengujian dianalisis dengan menggunakan API Web Software [7].

C. Hasil Isolasi Bakteri Asam Laktat yang Didapatkan

Sebanyak dua ose isolat BAL yang telah berumur 84 jam diinokulasikan ke dalam 5 ml
aquades steril. Selanjutnya suspense tersebut diinokulasikan sebanyak 2 ml ke dalam 5 ml
aquades steril hingga kekeruhan mencapai 2 McFarland. Sebanyak 4 ml larutan pada kekeruhan
2 McFarland diinokulasikan ke dalam ampul yang berisi medium API 50 CHL dan
dihomogenasi.
Sebanyak dua belas isolat BAL yaitu SKE1, SKE2, SKE3, SKE4, SKE5, SKE6, SKE7,
SKE8, SKE9, SKE10, SKE11, dan SKE12 diperoleh dari hasil isolasi. Isolat tersebut
membentuk zona bening pada media MRS agar yang mengandung CaCO3 1 %. Semua isolat
BAL yang diperoleh merupakan Gram positif dan katalase negatif. Isolat SKE5 memiliki
kemiripan dengan Genus Pediococcus karena memiliki bentuk sel tetrad dan memiliki koloni
bewarna putih. SKE 9 diketahui sebagai bakteri Lactobacillus curvatus.

Artikel II
Setyawardani, T, dkk, 2011. Identification and Characterization of Probiotic Lactic Acid
Bacteria Isolated from Indigenous Goat Milk. Journal of Animal Production, Volume 13,
Nomor 1, Halaman 57-63.
Impact Factor: 1.50

Hasil dari Isolasi Bakteri Asam Laktat


The isolation of LAB from the milk of Ettawa crossbreed (PE) resulted in 16 isolates had
been identified, The results of Gram coloring showed that 33 LAB isolates from the milks of PE
and PESA were positive Gram bacteria of rod shape (26 isolates), of short rod shape (1 isolate),
of round, oval shape (3 isolats), and of round shape (3 isolates) with the characteristic of negative
catalase. According to Salminen et al. (2004), LAB are positive Gram bacteria in the shapes of
rod or round and have the characteristic of negative catalase. All of the isolates in this study were
able to grow at 37oC (100%) and 24 isolates at 45oC (73 %), however, those that were able to
survive at 10oC were only 18 isolates (54,5%). The test of LAB tolerances toward salt
concentrations of 4 and 6.5 % showed that all isolates of LAB were capable of surviving for 7
days. All of the LAB isolates were able to survive at alkali pH (pH 9.6).
This study has successfully identified 16 LAB isolates from Ettawa crossbreed (PE) goat
and 17 isolates of LAB from the milk of Saanen crossbreed (PESA) goat. Physiological test
indicates that all LAB isolates have negative catalase characteristic, are able to grow at 37 and 24
isolates at 45oC (73%), however, there are only 18 isolates (54, 5%) that are able to survive at
10oC. All LAB isolates are able to survive up to 7 days in salt concentrations of 4 and 6.5 %,
survive at alkali pH (pH 9.6) and only 15 isolates (45%) that are capable of surviving at acidic
pH (pH 4.4). Twenty LAB isolates have the hetero-fermentative characteristic (61 %) and 13
isolates (39 %) are homo-fermentative.
All isolates do not produce dextran and NH3 from arginine and 18 isolates are able to
survive at pH 2.0, 2.5, and 3.2 with the decrease in the number of microbial colony of less than 1
log unit. As many as seven isolates are able to survive in 0.3% of bile salt, namely number 2, 3,
4, 10, 14, 26, and 28 with the decrease in the number of microbial colony, in the range of 1-2 log
units. The results of genus and species identifications by using API show that the potential
isolates as probiotic LAB are Lactobacillus rhamnosus (isolates of number 2 and 3),
Lactobacillus plantarum

Artikel III

Widodo, dkk, 2016. Isolation and Identification of Goat Milk-Derived Lactobacillus


Paracasei M104 and Pediococcus Pentosaceus M103 and Their Potential Use as Starter
Culture for Fermentation. Journal of Microbiology, Biotechnology and Food Sciences,
Volume 5, Nomor 4, Halaman 374-377.

Impact Factor: 0.9801

A. Metode Preparasi Susu Segar, Isolasi Bakteri dan Identifikasi Bakteri Asam Laktat

Fresh goat milk samples were obtained from three different farms in Yogyakarta, Indonesia.
Fresh milk was cooled immediately after milking in an ice box and transported to the laboratory
for analysis. An 1ml aliquot of fresh goat milk was added to 9 ml of 0.1% (w/v) sterile peptone
water to obtain 10-1 dilution. After three-fold serial dilution 0.1 ml aliquots were surface plated
on de Man Rogosa and Sharpe (MRS) agar (Merck) supplemented with 0.15% (w/v) ox bile then
incubated anaerobically at 37C for 48 hours. White colonies visible on the plate after incubation
were subjected to morphological and physiological analysis, including Gram staining, a catalase
test, assessments of shape, spore formation, motility and CO2 and NH3 production and
comparison of growth at 10C and 45C. These screening tests were used to select colonies with
LAB characteristics which were then subjected to molecular identification using 16S rRNA gene
amplification.

We obtained 4 isolates with the morphological and physiological characteristics of LAB


(Table 1). LAB are usually characterised as Gram-positive, aerobic or facultative anaerobic, non-
motile asporogenous rods and cocci with the ability to ferment carbohydrate with lactic acid as
the main fermentation product (Franz et al., 2010). Preliminary identification was based on
morphological and physiological analysis, ability to produce CO2, growth at different
temperatures (10C, 37C and 45C), pHs (4.4 and 9.6) and in media containing 6.5% NaCl.

Comparison of the fermentation patterns of all isolates with Axelssons (2002) classification
of LAB resulted in identification of three genera of LAB; M101 and M103 were identified as
Pediococci, M102 as Lactococcus and M104 as Lactobacillus (Table 1). All isolates were
homofermentative without CO2 production and isolates M101 and M103 were intolerant of salt
(NaCl 6.5%). 16S rRNA gene sequencing suggested that isolates M101 and M102 were close
homologues (97% similarity) of Lactococcus garvieae strain 29; isolate M103 was a close
homologue (96% similarity) of Pediococcus pentosaceus strain LAB6 and isolate M104 was a
close homologue (97% similarity) of Lactobacillus paracasei subsp. paracasei strain X212
(Figure 1).

Pustaka II Jenis Fungi yang Ditemukan di Susu Kambing

Sumber: 0.867

Vosgan, Zorica, dkk, 2015. Fungal Diversity in Goat Milk Obtained by Manual Milking. Journal
of Agroalimentary Process and Technologies. Volume 21 Nomor 3, Halaman 247-251.

Impact Factor: 1.30


The results of this study show the load and the diversity of fungi in the goat milk obtained by
hand milking. During grazing at higher altitudes (1000-1300 m), contamination of milk with
yeast and molds is higher, and also their incidence on food. The present study showed variations
in milk fungal communities according perioada de lactatie. It is important to respect the
minimum safety precautions in order to prevent spore germination and vegetative forms
formation that can produce toxic metabolites endangering consumer health. Fungal diversity of
the fresh goat milk was evidenced by direct microscopic examination; there were identified yeast
genera such as: Kluyveromyces sp., Rhodotorula sp., Cryptococcus sp. The appearance of
colonies and microscopic aspects of the main types of yeast identified in the goat milk are shown
in Figure 1.

Jurnal III Metode Pengujian Aktivitas Antijamur


Sumber:
Delavenne, J, dkk, 2012. Biodiversity of Antifungal Lactic Acid Bacteria Isolated froam Raw
Milk Sample from Cow, Ewe and Goat Over One-Year Period. International Journal of Food
Microbiology, Volume 155, Halaman 185-190.
Impact Factor: 3.339
2.1. Raw milk samples
Raw milk was sampled during year 2009 in refrigerated bulk tanks from milk collecting trucks
coming from restricted areas in three French Dpartements according to the three tested
species: ewe (Aveyron), goat (Deux Svres) and cow (Ille-et-Vilaine). For each species, nine raw
milk samples were collected during three successive weeks at three periods of the year (winter,
spring and summer/fall). They corresponded to January, April and July for ewes; February, June
and November for goats; and February, June and September for cows. These periods were
different depending on species; they corresponded to different lactation periods (beginning,
middle and end) for ewes and goats as well as different feeding conditions. In winter, ewes were
kept in sheep-folds and they were fed with dried food (grains and hay), they did not receive
silage. They started to graze outside in spring. Cows and goats were mainly kept in stalling
during winter and fed with dried and humid food (hay, silage and grains); they started to graze
outside in spring. Milk samples (500 ml) were aseptically taken in refrigeration tanks, kept at 4
C, sent to the laboratory and analyzed within 48 h. Milk pH was recorded and 10 ml were
immediately used for bacterial enumeration.

2.2. Enumeration of total and lactic acid bacteria


Raw milk samples (10 ml) were serially diluted 10-fold in peptone water (0.1%, pH 7) and plated
in quadruplicate on both non selective and semi-selective media. Total bacteria were enumerated
on plate count agar (PCA) supplemented with 1% milk (AES Chemunex, Bruz, France) (Michel
et al., 2001) and Elliker agar (Sigma-Aldrich, Saint-Quentin Fallavier, France) (Carr et al., 2002;
Randazzo et al., 2002; Teuber, 1993). Lactobacilli were enumerated on LAMVAB, an
MRSbased medium supplemented with vancomycin and acidified to pH 5 (Coeuret et al., 2003;
Hartemink et al., 1997; Henri-Dubernet et al., 2008). Enterococci, pediococci and lactobacilli
were enumerated on acidified MRS (pH 5.5) (Carr et al., 2002; Randazzo et al., 2002). The
kanamycin esculin azide agar medium (KEA, Sigma-Aldrich) was chosen for enterococci
(Franciosi et al., 2009; Teuber, 1993). M17 and a PCA based-medium enriched with 10% of
milk and supplemented with nalidixic acid (PCA-ATB) were used to enumerate lactococci
(Corroler et al., 1998; Franciosi et al., 2009). Leuconostoc strains were enumerated on Mayeux
Sandine and Elliker (MSE, AES Chemunex, Bruz) medium (Randazzo et al., 2002) and
psychrotolerant lactic acid bacteria enumerated on MRS (pH 6.2) at 10 C for 4 to 7 days
(Teuber, 1993). Fungi were enumerated on yeast extract glucose chloramphenicol medium (yeast
extract 0.5%; glucose 2%; chloramphenicol 100 mg/l) at 25 C (Casalta et al., 2009). Depending
on population tested, plates were incubated aerobically or in anaerobic jars at 10, 21, 30 or 37 C
for up to 7 days according to reference instructions. Cell counts were expressed as Log10
CFU/ml of milk. After enumeration, four plates of each dilution were overlaid with the four
tested fungi.

2.3. Preparation of yeast cell and mold spore suspensions


Four ubiquitous fungi, commonly encountered in cheese and yogurt spoilage (Caggia et al.,2001;
Hayaloglu and Kirbag, 2007; Wouters et al., 2002) were chosen as indicator microorganisms for
antifungal assays. The molds Penicillium expansum CBS 32.548NT and Mucor plumbeus CBS
129.41T came from the Culture Collection of Centraalbureau voor Schimmelcultures (CBS,
Utrecht, The Netherlands). The yeasts Pichia anomala LMSA 2.01.001 and Kluyveromyces
lactis CLIB196 came from the Souchothque de Bretagne culture collection (Universit de
Brest, Plouzan, France) and Collection de Levures d'Intrt Biotechnologique (INRA,
Thiverval-Grignon, France), respectively. Molds were cultivated on potato dextrose agar (PDA,
Difco, Le Pont de Claix, France) slants for a few days at 25 C until spores were formed. Spores
were then harvested with sterile distilled water supplemented with 0.1% Tween-80 and the
suspension was spread on PDA in a Roux flask to increase spore production (Roy et al., 1996).
Spores were harvested, enumerated using a Malassez cell and stock suspensions were
standardized to a final concentration of 107 spores/ml before storage at 80 C in glycerol (10%,
v/v). Before use, yeasts were subcultured in a yeast extract and malt based medium (YEMA)
composed of yeast extract (0.3%) and malt extract (2%) for 24 to 48 h at 25 C. One hundred
microliters of this suspension were inoculated in 10 ml of YEMA broth and incubated
aerobically at 25 C for 24 h. Cells were enumerated with a Malassez cell and the resulting
suspension was standardized at a concentration of 106 cells/ml in peptone water (0.1%). Yeast
stocks were maintained at 80 C in YEMA supplemented with glycerol (30%, v/v).

2.4. Antifungal activity assays


After enumeration of LAB, agar plates used for bacterial enumeration (except for the non
selective PCA medium) with less than 300 colonies were overlaid with 8 ml of PDA for molds or
YEMA medium for yeasts (agar 0.8%) containing 104 fungal spores or yeast cells per ml. Plates
were then incubated at 25 C and areas of inhibition recorded after 48 h (Magnusson and
Schnrer, 2001). Active colonies showing clear inhibition area, were isolated on appropriated
growth medium supplemented with amphotericin (5.6 mg/ml) (Sigma- Aldrich), followed by a
minimum of three successive subcultures. A second antifungal activity assay was performed on
purified isolates to confirm the first test. Active isolateswere maintained in growth medium
supplemented with glycerol (30%, v/v) at 80 C. 186 .

2.5. Identification of antifungal isolates


Isolates were examined by phase-contrast microscopy, Gram stained and tested for the presence
of catalase. For isolate identification, total DNA was extracted from 1.5 ml of pure culture
obtained after 18 h incubation in MRS broth as described previously by Randazzo et al. (2002),
and used as template for the 16S rRNA gene amplification with the pA/pH bacterial universal
primers pair (Edwards et al., 1989; Zamfir et al., 2006). PCR amplicons were sequenced at the
Biogenouest sequencing platform in the Station Biologique de Roscoff (http://www.sb-
roscoff.fr/SG/) and sequences were aligned on the NCBI website (http://www.ncbi.nlm.nih.gov/
BLAST) (October 2010) for species assignment.

2.6. Statistical analyses


Statistical analyses were performed using STATGRAPHICS Plus 5.1 package (Statistical
Graphics Corp., VA, USA). A three-way analysis of variance (ANOVA) was used to compare
microbial counts on agar media and to assess the effects of animal species (A: cow, goat or ewe),
periods of sampling (P: first, second or third), enumeration media and interactions between them.
When significant effects were found, individual means were compared by one-way ANOVA
using the Tukey's honestly significant difference (HSD) test. Statistical significance was set at
Pb0.05 level. Microbial counts on enumeration agar were expressed as means (Log10 CFU/ml)
of four replicates. Dependences between numbers of antifungal isolates were obtained by Chi
Square test of independence under Microsoft Office Excel 2003 software (Microsoft Office
Professional Edition 2003). Statistical significance was set at Pb0.05 level for this test. Animals,
periods of sampling, media, dilutions, fungal targets and taxa were considered as variables.

2.7. Lactobacilli biodiversity analyses


The diversity of lactobacilli was analyzed according to animals and sampling periods, by
calculating the ratio (pi) between number of antifungal Lactobacillus belonging to phylogenetic
group i (ni) and the total number of lactobacilli (N): pi=ni/N, for each animal-sampling period
couple. Diversity was measured by the Shannon index (H) (Shannon and Weaver, 1963) using
the following equation: H= pi Log2 (pi). The higher the H index is, the greater the diversity.
Each animal-sampling period couple containing different numbers of Lactobacillus groups, the
diversity within each animal-sampling period couple was also compared by calculating the
equitability (E=H/log2 [S], with S being the number of Lactobacillus groups). The equitability
can vary from 0 to 1, an E value equal to 0 means that one species clearly dominates among
others, whereas an E value close to 1 indicates that individuals from each group have an
equitable distribution.