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Perspective on Testing Methods–Challenges and Opportunities in

the Beverage Category

Former Chief Scientist-CSIR-CFTRI, MYSORE 570020

Beverage Sampling & Testing Methodology, 25th February, 2019, New Delhi

Food Analysis: The Basis

Total sugarAll mono-and disaccharides present in food, derived from any source. In practice, this primarily consists of sucrose(table sugar), fructose, glucose (dextrose and lactose (milk sugar).”Sugar” usually refers specifically to sucrose (table sugar).

Added SugarSugars added to foods during processing or preparation (e.g., brown sugar, corn sweetener, corn syrup, dextrose, fructose. Sucrose, high-fructose corn syrup, honey, invert sugar, lactose, maltose, malt syrup, molasses, raw sugar. Added sugar excludes naturally occurring sugars present in the fruit, vegetable, or dairy products or in juiced fruits and vegetables

Free SugarFree sugar includes all sugars whether naturally present or added by the manufacturer to the juice or beverage

Food and Nutrition Sciences, 2012, 3, 1509-1513

• Invert sugar: Partially inverted sucrose, wherein about one-half of the sucrose has been hydrolyzed to glucose and fructose. This ratio of approximately 1:1:2 (glucose: fructose: sucrose) closely matches the ratio found in orange juice.

• Molasses

Rice, Potato, Sugar-beet

Maize, Sorghum, Sugarcane and Millets

Pineapple,

13C/12 C RatioC4 plants: -16 to -10 ‰CAM plants: -20 to -10 ‰C3 plants: -33 to -24 ‰

Element Isotope Relative abundance(%)

Hydrogen

1H 99.98852H (D) 0.0145

Carbon

12C 98.89213C 1.108

Nitrogen

14N 99.633715N 0.3663

Oxygen

16O 99.758617O 0.037518O 0.2038

The measurement of the ration (X heavy/X common) gives information about botanical (13C/12C), animal (15N/14N) or geographic origin (2H/1H, 18O/16O)

Fruit juice and beverages• Sweetening (13C/12C ratio)• Watering (2H(D)/H and 18O)

• When cane sugar is the source of inverted sucrose, SIRA can be used based as ratio of 13C /12C

• Beets, have a 13C/12C about the same for sugars in orange juice. It is inadequate for detecting adulteration by beet sugar.

• Water in orange juice has higher deuterium (2H) and 18O concentrations than sugar beet invert syrup water.

• Detection method is based on D/H and 180/O16 ratios in sucrose of orange juice

Fruit juice with no added water is preferential to juice from concentrate.18O and 2H isotope analysis allows detection of added water18O determination of water in fruit juice is widely used to establish whether a juice has been reconstituted from concentrate using local water.

AcaiAppleApricotAroniaBananaBlackberryBlueberryBoysenberryCarrotCherryCoconutCranberryCurrantGoji BerryGrapeGrapefruitGuavaKiwiLemonLime

LingonberryLycheeOrangeMangoMangosteenMelonPapayaPassion FruitPeachPearPineapplePlumPomegranatePruneRaspberryStrawberryTomatoTamarindYum berry

AcaiGoji berryMangosteen

Rambutan

Dragon fruit

Paw paw Guava

Passion fruit

Cranberry

Anthocyanin profiling to detect juice adulteration

• Pattern of anthocyanin profile by RP-HPLC is often characteristic of a fruit type

ElderberryCranberry

13

Cranberry anthocyanin profile

min2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

DAD1 A, Sig=520,4 Ref=590,20 (ANTH1403\MACRA.D)

Cyanidin-3-galactoside

Cyanidin-3-arabinoside

Peonidin-3-galactoside

Peonidin-3-glucoside

Peonidin-3-arabinoside

min2 4 6 8 10 12 14 16 18

mAU

0

10

20

30

40

Additional peaks

Peak too strong

relative to other

anthocyanins for

cranberry Elderberry addition

• What does food do for me?

– Nutritional optimization of the quality of life

– Identification of physiological active components to prevent or delay premature onset of chronic disease

• Nutraceuticals• Phytochemicals• Pre/Probiotics• Fiber

Era of Nutraceuticals & Functional Foods

Changing Nutrition/Health Scenario:

Functional Foods & Beverages

Vitamins MineralsPlant/Herbal extractsEnzymesProteins/peptidesPro- and pre-bioticsAmino acidsNucleotidesPolyphenols

Breakfast on the goProbiotic fortified drinksOmega-3 enriched drinksHigh energy drinksSports drinksSlimming drinksFortified fruit juicesFortified waterAntihangover drinks

Only as examples, taken at random, speaker does not endorse or own these in any way.

Vitamins16

Minerals 15

Amino acids & other nutrients

25

Nucleotides 4

Probiotics28

Prebiotics13

Plant/Botanical Ingredients

400NutraceuticalIngredients

221As listed in Schedules 1, II, IV, VI, VII, and VIII

1. Fit for purpose methods2. Validated method to be verified3. Standards

I. Vitamins (water and Fat soluble)II. SugarsIII.SweetenersIV. Minerals (Na, K, Ca, MgV. GinsenosidesVI. CaffeineVII.Biomarker for guarana extractVIII.Epigallocatechin

4. LC-MS/MS, ICP-MS5. HPLC with RI/ELSD detector6. Qualified and competent manpower

• Method must accurately quantify the compound of interest.

• Time consuming and expensive, both in terms of man-hour and equipment/consumables costs.. The validated final method can be specific for single or multiple matrices, but each matrix must be tested as part of the method development and validation exercise.

• Before the method can be put into routine use, the validation report must be issued and approved.

• Subsequently, the method can be transferred to the laboratory where appropriate training of personnel can be undertaken.

• When the required level of proficiency is attained, the method can be placed into routine use.

FSSA(I) Regulation:The viable number of organisms in food with added probiotic ingredients shall be 108 CFU/g.

No Organism No Organism

1 Lactobacillus acidophilus 15 Lactobacillus amylovorus

2 Lactobacillus plantarum 16 Lactobacillus gallinarum

3 Lactobacillus reuteri 17 Lactobacillus delbrueckii

4 Lactobacillus rhamnosus 18 Bifidobacterium bifidum

5 Lactobacillus salivarius 19 Bifidobacterium lactis

6 Lactobacillus casei 20 Bifidobacterium breve

7 Lactobacillus brevi 21 Bifidobacterium longum

8 Lactobacillus johnsonii 22 Bifidobacterium animalis

9 Bacillus coagulans 23 Bifidobacterium infantis

10 Lactobacillus fermentum 24 Streptococcus thermophilus

11 Lactobacillus caucasicus 25 Saccharomyces boulardii

12 Lactobacillus helveticus 26 Saccharomyces cerevisiae

13 Lactobacillus lactis 27 Lactobacillus paracasei

14 Lactobacillus delbrueckiisub- sp. bulgaricus

28 Lactobacillus gasseri

Do we have Reference cultures?The precise identification of these bacteria to the species level is not an easy task.1. Identification of Lactobacillus isolates by

phenotypic methods is difficult because it requires, determination of bacterial properties beyond those of the common fermentation tests.

2. In general, about 17 phenotypic tests are required to identify an isolate of Lactobacillusaccurately at the species level.

3. DNA fingerprints generated by restriction endonucleases.

4. Amplified ribosomal DNA restriction analysis (ARDRA)

Beverage analysis is continuously demanding thedevelopment of more robust, efficient, sensitive, andcost-effective analytical methodologies to guaranteethe quality, safety, quality, and traceability during theshelf life of products, in agreement with consumerdemands and legislation requirements.

Consequently, research about novel technologies usedin beverage analysis that allow food analysts tosimplify sample preparation, and increase speed andsensitivity in the analysis of ingredients, additives,flavors, and food contaminants, is a promisingchallenge for future trends.