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GB 4789.16-2016: Microbiological examination of food hygiene -- Identification of common mycotoxin-producing fungi
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Basic data | Standard ID | GB 4789.16-2016 (GB4789.16-2016) | | Description (Translated English) | Microbiological examination of food hygiene -- Identification of common mycotoxin-producing fungi | | Sector / Industry | National Standard | | Classification of Chinese Standard | C53 | | Word Count Estimation | 31,339 | | Date of Issue | 2016-12-23 | | Date of Implementation | 2017-06-23 | | Older Standard (superseded by this standard) | GB/T 4789.16-2003 | | Regulation (derived from) | National Health and Family Planning Commission Notice No.17 of 2016 | | Issuing agency(ies) | National Health and Family Planning Commission of the People's Republic of China, State Food and Drug Administration | GB 4789.16-2016: Microbiological examination of food hygiene -- Identification of common mycotoxin-producing fungi
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Microbiological examination of food hygiene - Identification of common mycotoxin-producing fungi
National Standards of People's Republic of China
National Food Safety Standard
Food Microbiology Inspection
Morphological Identification of common mycotoxin
Issued on. 2016-12-23
2017-06-23 implementation
National Health and Family Planning Commission People's Republic of China
China Food and Drug Administration released
Foreword
This standard replaces GB/T 4789.16-2003 "Microbiological examination of food hygiene common mycotoxin producing identification"
This standard compared with GB/T 4789.16-2003, main changes are as follows.
--- Standard name was changed to "national food safety standards of food microbiology testing common mycotoxin producing morphological identification";
--- Increased inspection procedures;
--- Increased toxigenic species of Aspergillus niger, Aspergillus black, stick Aspergillus, Monascus, etc;
--- Modify the standard name;
--- Modify the equipment and materials;
--- Modify the media and reagents;
--- Modify the morphological description of each species;
--- Revised Appendix A;
--- Remove the yellow green green mold, Penicillium Island, wrinkles Penicillium, Penicillium produce purple, red and other Penicillium species;
--- Delete keys original Appendix B, Appendix C, Appendix D.
National Food Safety Standard
Food Microbiology Inspection
Morphological Identification of common mycotoxin
1 Scope
This standard specifies the method of identifying common toxigenic fungi in food.
This standard applies to Aspergillus (Aspergillus), Penicillium (Penicillium), Fusarium (Fusarium) and other species often
See the identification of toxigenic fungi.
2 Equipment and Materials
In addition to the microbiological laboratory conventional sterilization and cultivation equipment, other equipment and materials as follows.
2.1 Refrigerator. 2 ℃ ~ 5 ℃.
2.2 incubator. 25 ℃ ± 1 ℃.
Microscope 2.3. 10 to 100 times.
2.4 ocular micrometer.
2.5 lens micrometer.
2.6 biological safety cabinet.
2.7 thermostatic water bath.
2.8 inoculation hook.
2.9 Separation needle.
2.10 slide.
2.11 coverslip.
2.12 stainless steel knife or ophthalmic surgical knife.
3 Reagents and culture medium
3.1 lactic acid phenol solution. see A.1.
3.2 Czapek medium. see A.2.
3.3 potato - dextrose agar medium. see A.3.
3.4 malt extract agar medium. see A.4.
3.5 sugarless potato agar medium. see A.5.
4 steps
4.1 colony characteristics observed. In order to develop a complete record of the colony for observation may be pure cultures o'clock on the plate. Aspergillus, Penicillium usually
Czapek medium was inoculated with Fusarium inoculation usually takes a variety of media, other fungi commonly used potato - dextrose agar medium.
The plates were inverted, up inoculation point or three points, each strain was inoculated two plates, is placed in 25 ℃ ± 1 ℃ incubator conducted training
support. When small colonies just grow, remove a plate aseptically with sterile ophthalmic surgical knife stainless steel knife or colonies, along with culture
Quiche under 1cm × 2cm pieces, the home side colony, cultured, observed at 5d ~ 14d. This culture method instead of a small, significant
Fruiting bodies borne police state.
4.2 ramp observation. The fungal pure cultures streaked (Aspergillus, Penicillium) or point species (Fusarium or bacteria) to the plane, train 5d ~ 14d,
Observe colony morphology, but also can directly observe the shape and arrangement of the spores under low magnification microscope tube set slant.
4.3 Producer. Take a glass slide add a drop of lactic acid phenol solution, take a small hook inoculation fungal cultures, set lactic acid - phenol solution, with two separate
Needle culture was gently torn to pieces, should not apply, so as not to destroy the structure of the fungus. Then add coverslip, if bubbles can be heated in the alcohol lamp
exclude. Production should be in a biological safety cabinet or a sterile hood vaccination or inoculation box or glove box operation to prevent spores flying.
4.4 Microscopy. morphology was observed fungal hyphae and spores, features, spores, etc. are arranged, and recorded.
5 test procedure
Common mycotoxin producing morphological identification test program shown in Figure 1.
1 common mycotoxin producing morphological identification of inspection procedure
6 genera and morphological characteristics of fungi may produce mycotoxins
6.1 Aspergillus (Aspergillus)
Toxigenic fungi of the genus include Aspergillus flavus, Aspergillus parasiticus, Aspergillus versicolor, Aspergillus nidulans, Aspergillus ochraceus, Aspergillus niger, Aspergillus and black curved rod
Mold and so on. These fungi may produce secondary metabolites of Aspergillus flavus toxin, ochratoxin, fumonisin, patulin and so on.
Aspergillus mycelium colorless, transparent or in bright colors, but not dirt color was dark; fertile conidia stalks in a generally perpendicular direction from
Specialized foot thick-walled cells give birth, smooth or rough, usually without diaphragm; forming a top top inflated balloon, with different shapes, formed from the surface of the bottle
Stems, or to produce stem base, and then forming a bottle stems from the stem base, and finally produce spores stems from the bottle. Conidia unicellular, with a variety of different shapes and
Color, smooth or ornamented, connected unbranched chains. From the top of the capsule to conidia chains of different shapes conidia head, revealing different
Colour. Some species can form a thick shell cell shape varies by species; some species can be formed sclerotia or sclerotia class structure; there are species produce
Sexual stage, to form a closed capsule shell containing asci and ascospores, ascospores mostly transparent or with different colors, shapes and ornamentation.
6.1.1 flavus (A.flavus)
Colonies grew rapidly in Czapek agar, 25 ℃ 7d diameter 35mm ~ 40mm (~ 70mm), 12d ~ 14d of 55mm ~
70mm; velvet-like texture is mainly dense, sometimes a little now or a central portion of fluff were flocculent, or are flat to irregular radial grooves; points
Endospores more structure, the color is yellow-green to green grass, early lighter, slightly darker, mostly in shallow water near the green after Qin old (LightCressGreen →
CressGreen, R.XXXI), there was a green Osmanthus (MignonetteGreen, R.XXXI), dark green grass (Kronberg'sGreen,
R.XXXI) or emerald green (JadeGreen, R.XXXI) were initially some strains are now yellow, nearly strontium yellow (StrontiumYelow, R.
XVI), then green; generally no exudate; some strains formed small or large sclerotia, a lot of time, affecting the appearance of the colony accompanied by exudate,
Colorless to light brown, colony reverse colorless to pale brown, sclerotia-producing strain in the back at the sclerotia appear dark brown spots. Meristem
First spores spherical head, after radiate, (80 ~) 200 ~ 500 (~ 800) μm, or breaks into a few loose columnar body, there are a few were short column
Like those; conidiophores mostly students from the matrix, spore stalks (200 ~) 400 ~ 800 (~ 3000) μm × (4 ~) 9.6 ~ 16 (~ 20) μm, wall
Thick, colorless, rough to very rough; top to the flask-shaped capsule nearly spherical diameter (9 ~) 23 ~ 50 (~ 65) μm, most fertile surfaces, only the upper part of the small
Fertile; sporulation bilayer structure. terrier group 6.2 ~ 13.2 (19 ~) × 3.2μm × 6μm, bottles Terrier 6.2 ~ 12μm × 2.4 ~ 4μm, some small top
Bottle capsule had only stems; conidia mostly spherical or near spherical (2.4 ~) 3.6 ~ 4.8 (~ 6.4) μm, few oval 3.2 ~ 5.2μm ×
2.7 ~ 4.2μm, slightly rough to the wall with small spines; some strains produce sclerotia, white at the beginning, after the old brownish black, spherical or near spherical shape, size or
Number of different, generally (280 ~) 420 ~ 980μm, shown in Figure 2.
Some strains of Aspergillus flavus produces aflatoxin (Aflatoxins).
Figure 2 Aspergillus flavus spore structures and conidia
6.1.2 Aspergillus parasiticus (A.parasiticus)
Colonies on Czapek agar 25 ℃ 7d diameter 35mm, 12d ~ 14d50mm ~ 60mm; velvet-like texture, occasional floc bacteria
Silk, margin white; spores more structure, the color is dark green, near Cedar Green (CedarGreen, R.VI), after nearly darken old ivy
Green (IvyGreen, R.XXXI); there are radial grooves, no effusion, negative colonies pale brown, nearly grayish brown (LightDrab, R.
VLVI). First conidia spherical head, after radiate diameter of 84μm ~ 210μm; Conidiophore born from the matrix, spore stalks 140 ~
420 (~ 800) μm × 6 ~ 12μm, wall rough now, there are nearly smooth; top sac pestle-shaped or flask-shaped, diameter (13.8 ~) 17 ~
31μm, most fertile surface; sporulation structure monolayer bottles Terrier 6.2 ~ 10.6μm × 2.4 ~ 4μm, occasionally observed is longer reachable
15μm, with a middle wall; spores spherical or near spherical, (3.6 ~) 4.2 ~ 5.5 (~ 6.4) μm, the wall is slightly rough, with crude sulfur small spines; not
See sclerotia, shown in Figure 3.
Aspergillus parasiticus strains can produce aflatoxin.
Figure 3 Aspergillus parasiticus spores and spore structures
6.1.3 Aspergillus versicolor (A.versicolor)
Limitations colonies grown on Czapek agar, 25 ℃ 7d diameter 15mm ~ 17mm, 10d ~ 12d of 24mm ~ 30mm; texture
A velvet-like or flocculent, or coexist with each other or both texture mosaic; colonies uneven, thicker central part of the uplift or depression, or in varying degrees
Warping; color vary widely, early white, later showed different degrees of green, close to the green beans, green flower Fu (PoisGreen, Asphodel
Green, R.XLI) deep green grapes (DeepGrapeGreen, R.XLI), some strains were malachite green (MalachiteGreen, R.XXX
Ⅱ), olive green (OliveGreen, R.Ⅳ) or dark olive-brown (DarkOliveBuff, R.XL), sometimes there are those who are not green, depending on points
How many students spore structures; mycelium lanes brown, pink, light brown close to pink (BuffyPink, R.XXLⅢ) or pale pink cinnamon
(PalePinkishCinnamrn, R.XXIX), because different strains varies; sometimes forming spores structure slow; there are radial grooves; infiltration
With or without a liquid, nearly colorless or light brown to purple to form droplets or gather into large drops; no smell or slightly musty; colony reverse Yen
Color varies, nearly colorless to pale brown, pink, purple or purple-brown rose, the spread of pigment in the matrix; some strains of cells to form a shell, the old culture
Yang was visible clumps together into a pale yellow. Conidial head is small, spherical beginning after radiate in diameter (25 ~) 75 ~ 125μm; points
Spore stalk stem directly from the matrix by students (45 ~) 150 ~ 300 (~ 500) μm × 4 ~ 8μm, born from aerial hyphae were mostly very short, colorless or slightly
With yellow, thick-walled, smooth; vesicle hemispherical, slightly longer or slightly oval, diameter 9 ~ 20μm, about 3/4 of the fertile surface, only a small vesicle
Top conidia stalks slightly inflated without significantly; sporulation bilayer structure. terrier group generally 5 ~ 8μm × 2 ~ 3μm, bottle Terrier 6 ~ 8μm × 1.5
~ 2.5μm; spores spherical or near spherical, green, diameter (2 ~) 2.5 ~ 3.5 (~ 4) μm, rough walls, with small spines; or near-spherical shell cell
Spherical diameter of 11μm ~ 20μm, formed together into a large number of yellow clumps Figure 4.
Certain strains of Aspergillus versicolor can produce sterigmatocystin (sterigmatocystin).
Explanation.
1,4 --- sporulation structures;
2 --- incomplete sporulation structures;
3,5 --- conidia;
6 --- shell cells.
Figure 4 Aspergillus versicolor sporulation structures housing cells and spores
6.1.4 nidulans Emericella (Emericellanidulans)
Anamorph. nidulans (A.nidulans)
Colonies on Czapek agar 25 ℃ 7d diameter of about 25mm, 12d ~ 14d45mm ~ 65mm; velvet-like texture or flocculent, flat
Tan or with obscure radiation groove; a large number of spores structure or less, showed different degrees of green, close to the emerald green to green cress (Jade
Green → CressGreen, R.XXXI) or Lincoln green to green flower Fu (LincolnGreen → AsphodelGreen, R.XLI) and even
Pale yellow olive (LightYelowishOlive, R.XXX); some cleistothecia scattered in the meantime, some strains produce large amounts of cleistothecia by
Shell surrounded by cells, so that the surface appears yellow or floc particles agglomerate structure, the colonies with brown, nearly deer brown, light brown or bronze
(FawnColor, BuffyBrown, R.XL; IsabelaColor, R.XXX); exudate more, or no less, pale reddish brown to purple-brown; no
Odor; colony reverse purple brown to purple-brown, brown deer near wine (VinaceousFawn, R.XL), slightly diffused in the matrix. Closed capsule
Shell spherical, dark purple, diameter of 100 ~ 250 (~ 300) μm, outside and showed yellow cells surrounded by a shell; ASCUS nearly spherical, 8 ~
12μm, generally mature in two weeks, while others are immature; ascospores lenticular-shaped, 3.5 ~ 5μm × 3.2 ~ 4μm, with two complete
Equatorial crown, with pleats, width of 0.5 ~ 1μm, purple, smooth convex surface. Conidia early to head for the radial spherical diameter of 30 ~ 70μm, old
After the short form dense cylindrical, 50 ~ 100μm × 30 ~ 40μm; Conidiophore born from the matrix or aerial hyphae, spore stalks are mostly short (~ 20)
50 ~ 150 (~ 300) μm × 3.5 ~ 6.5μm, slightly curved, with dark brown, smooth wall; hemispherical top bag, the same color and spore stalk stem diameter
(5.5 ~) 8 ~ 16μm, the upper half of the surface of fertility; sporulation bilayer structure. terrier group 4 ~ 8μm × 2.5 ~ 3μm, some abnormal strain on the top of the capsule
Hypertrophy formed nearly spherical terrier group, bottle Terrier 5 ~ 8μm × 2 ~ 2.5μm; spores spherical (2.5 ~) 3 ~ 4 (~ 4.5) μm, rough,
Occasionally nearly smooth; cells mostly spherical shell diameter of 11μm ~ 25μm, few longish or oval 18 ~ 40μm × 12 ~ 24μm,
See Figure 5.
Some strains of A. nidulans can generate sterigmatocystin.
Explanation.
1 --- sporulation structures;
2 --- conidia;
3 --- ascospores;
4 --- shell cells.
Sporulation Chart 5 nidulans of Emericella
6.1.5 OTA (A.ochraceus)
Colonies on Czapek agar 25 ℃ 7d diameter 25mm ~ 35mm, 10d ~ 12d of 35mm ~ 55mm; velvet-like texture or
Now a little fluff, flat or with inconspicuous radial grooves; conidia structure dense or sparse, light brown, buckskin color to near cinnamon
(Chamois, R.XXX → Cinnamon, R.XXIX) or yellow ocher (Yelow Ocher, R.XV), there are nearly in bronze
(Isabela, R.XXX) who mycelium white; sometimes forming colorless to brown exudate; slightly musty; some strains can form sclerotia, single
Alone or in groups; reverse colorless colonies showed different degrees of green or brown to purple-brown. First conidial head is spherical 75μm ~ 200μm, old
After up to 500μm, often split into several diverging dense columnar body; conidia stems mostly students from the matrix, spore stalks generally 500 ~
1500μm × (6 ~) 10 ~ 15μm, a small number of the elderly up to 200μm or more, mostly brown, wall thickness 0.7 ~ 1.5μm, rough; spherical vesicle
Or nearly spherical, (20 ~) 30 ~ 45 (~ 55) μm, the entire surface of fertile, with occasional small vesicle exists, only the top fertile; sporulation bilayer structure. terrier group
(7 ~) 10 ~ 25μm × (2.5 ~) 3 ~ 6μm, bottles Terrier 7 ~ 13μm × 1.5 ~ 3μm; spores are mostly spherical or near spherical diameter
(2 ~) 2.5 ~ 3.5 (~ 4) μm, few broadly elliptic, 3 ~ 3.5μm × 2.5 ~ 3μm, wall nearly smooth or rough fine. If the sclerotia
Spherical, oval or slightly longer, 500μm ~ 1000μm, early white, later purple-brown, shown in Figure 6.
Some strains can produce ochratoxin ochratoxin (Ochratoxins).
Figure 6 ochratoxin structure of sporulation and conidia
6.1.6 Aspergillus niger (A.niger)
Colonies grew rapidly in Czapek agar, 25 ℃ 7d diameter is generally 50mm ~ 70mm, a few strains more limited; flat or slightly Centre
Projections, regular or irregular radial grooves; or velvet-like texture is slightly flocculent, some strains sometimes infertility overgrowth; conidia
A large number of structures, the surface was dark brown to black carbon black; exudate with or without colorless; with or without musty; some strains produce sclerotia, the slant culture
Many born when support base; colony reverse colorless or slightly varying degrees of yellow, brown or yellowish green belt. First conidia spherical head to radiation
Shot shape, the diameter of 150 ~ 500μm, after the old split into several cylindrical structure, up to 800μm; Conidiophore occurs in the matrix, spore stalks
(800) 1500 ~ 3500 (~ 4000) μm × 9 ~ 20μm, wall smooth, with yellow or brown when old; vesicle spherical or near spherical diameter
(30 ~) 40 ~ 70 (~ 80) μm, brown when old, the entire surface of fertile; sporulation bilayer structure. Terrier group within a certain time frame with the strain age increase
Large increases, (10 ~) 15 ~ 35 (~ 70) μm × (3 ~) 5 ~ 8 (~ 14) μm, when old dark brown; phialides (7 ~) 8 ~ 10 (~ 12) μm ×
(2-3) after 2.5 ~ 3μm, spores spherical, nearly spherical or old becomes flat transverse diameter of 3 ~ 4.8 (~ 5.4) μm, with sharp or rough wall significantly
Warts, irregular ridges or vertical stripes, sometimes a little rough or nearly smooth now are; if sclerotia was spherical or near spherical diameter
700μm ~ 1000μm, or greater, cream to pale yellow, shown in Figure 7.
Certain strains of Aspergillus niger produces fumonisin B group (FumonisinsB).
Figure 7 Aspergillus niger spores and spore structures
6.1.7 Carbon black niger (A.carbonarius)
Colonies grew rapidly in Czapek agar, 25 ℃ 7d diameter is generally 40mm ~ 60mm, flat; velvet-like texture, and some strains
More aerial mycelium; conidia structure a lot, sometimes at the edges or near the edge of the small, pure black spore head; a small amount of a colorless exudate
Or no; inconspicuous slightly musty; colony reverse pale yellow to brown or yellowish brown to dark brown was central. First conidial head is spherical,
Diameter 100 ~ 300 (~ 500) μm, when the old split into several cylindrical structures, with diameters up to 1000μm or larger; Conidiophore occurred in
Matrix, spore stalks 2000 ~ 4500 × 20 ~ 30 (~ 40) μm, colorless and transparent, when the old part of the bag near the top with a yellow or light brown, smooth wall; top
Spherical or near spherical capsule diameter of 70 ~ 80 (~ 120) μm, old brown belt, the entire surface of fertile; sporulation bilayer structure. Terrier group at a certain time
With the range of strain age increases, generally 20 ~ 50μm × (5 ~) 7 ~ 13μm, and the larger up to 80μm, when old brown; bottle Terrier
8 ~ 12 (~ 15) μm × 3 ~ 6 (~ 8) μm; spores spherical, nearly spherical, diameter 6μm ~ 9μm, wall significantly rough, old-peaked after warty
Or warty, shown in Figure 8. Reported occasionally produce sclerotia.
Some strains can produce carbon black Aspergillus fumonisin and ochratoxin.
8 black Aspergillus spores and spore structures
6.1.8 rods Aspergillus (A.clavatus)
Colonies on Czapek agar 25 ℃ 7d diameter 23mm ~ 30mm, 12d up to 30 ~ 38 (~ 60) mm; velvet-like texture to powder
Like, annular and radial grooves; white mycelium, conidia structure dark blue-green, green to near Ai lily green (ArtemesiaGreen
→ LilyGreen, R.XLVII), sometimes conidia district is now part of yellow; with or without exudate, colorless or pale brown; no gas
Taste, and some strains are a strong rancid; colony reverse colorless or pale yellow. Conidia head childhood as a rod, a length of up to 300μm, straight
Diameter of up to 150μm, after the old fragmented into several compact cylinder; conidiophores occurs in the matrix, spore stalks of varying lengths, the shorter 300μm ~
400μm, the elderly up to 3000μm, diameter 20 ~ 30 (~ 50) μm, thin walls, smooth and colorless; top balloon inflated by a spore stalks gradually to the top
Is shaped sticks, lengths of up to 250μm, diameter 50 ~ 60 (~ 100) μm; sporulation single structure. the entire surface of the bottle stems densely born in the top of the balloon,
Generally 8 ~ 12μm × 2 ~ 3μm, who was born in the top of the capsule base relatively short; conidia elliptical, 3 ~ 4.5 (~ 5) μm × 2.5 ~
3μm, wall smooth; no sclerotia and sexual stage, shown in Figure 9.
Certain strains of Aspergillus rods can produce patulin (Patulin).
Explanation.
1 --- conidiophores and conidia head;
2 --- sporulation structures;
3 --- conidia.
9 bar niger spores and spore structures
6.2 Penicillium (Penicillium)
The genus toxigenic fungi, including Penicillium orange, orange gray Penicillium (Synonyms. Penicillium P.cyclopium), sallow Penicillium (Synonyms. Green Show
Mold P.patulum, nettle Penicillium P.urticae), bright green Penicillium (formerly. pure green Penicillium P.viridicatum). These fungi may produce
Secondary metabolites born citrinin, arc azo acid, patulin and so on.
Mycelium fine, with diaphragm, colorless or pale, there were less color, but will not have a dark, expand and produce a large number of irregular branching shape
Into different degrees of dense mycelium; colony by the edge of the mycelium composed generally clear, neat, little irregular persons; Conidiophore occur
In ambush type hyphae, hyphae substrate surface or aerial mycelium; spore stalks smaller, often with the diaphragm, some species showed varying degrees of expansion at its top
Great, produce or broom-like branches at the top of the top, smooth wall or exhibit different degrees of roughness; wherein the shape and complexity of the broom-like sticks is to identify points
The primary standard classes, broom-like branches whorled a single, two-wheeled students, three students, four students and irregular persons; producing stem cells have been produced bottles, close to each other,
Not close or nearly parallel, bottled, lanceolate, cylindrical and subterete are less, usually straight rather tha...
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